Apparatus for improved luminescence assays

ABSTRACT

An apparatus for performing a binding assay for an analyte of interest present in a sample based upon measurement of electrochemiluminescence at an electrode surface comprising a cell defining a sample containing volume intersecting with inlet and outlet means, an electrode having a substantially horizontally positioned surface exposed to and positioned below a portion of the sample containing volume, means for impressing electrochemical energy upon said electrode sufficient to generate luminescence, means for magnetically collecting particles along said surface and means for measuring the luminescence generated at said electrode.

This application is a division of application Ser. No. 08/346,832 filedNov. 30, 1994, which is a continuation of prior application Ser. No.08/158,193, filed Nov. 24, 1993 now abandoned, which in turn was acontinuation of prior application Ser. No. 07/652,427 filed Feb. 6, 1991now abandoned, which is a continuation in part of application Ser. No.07/539,389, filed Jun. 18, 1990 now abandoned, which in turn is acontinuation of application Ser. No. 07/266,882, filed Nov. 3, 1988 nowabandoned.

FIELD OF THE INVENTION

This application relates generally to methods and apparatus forconducting binding assays, more particularly to those which measure thepresence of an analyte of interest by measuring luminescence emitted byone or more labeled components of the assay system. More specifically,the invention relates to precise, reproducible, accurate homogeneous orheterogeneous specific binding assays of improved sensitivity in whichthe luminescent component is concentrated in the assay composition andcollected on the detection system before being caused toelectrochemiluminescence.

BACKGROUND OF THE INVENTION

Numerous methods and systems have been developed for the detection andquantitation of analytes of interest in biochemical and biologicalsubstances. Methods and systems which are capable of measuring traceamounts of microorganisms, pharmaceuticals, hormones, viruses,antibodies, nucleic acids and other proteins are of great value toresearchers and clinicians.

A very substantial body of art has been developed based upon the wellknown binding reactions, e.g., antigen-antibody reactions, nucleic acidhybridization techniques, and protein-ligand systems. The high degree ofspecificity in many biochemical and biological binding systems has ledto many assay methods and systems of value in research and diagnostics.Typically, the existence of an analyte of interest is indicated by thepresence or absence of an observable "label" attached to one or more ofthe binding materials. Of particular interest are labels which can bemade to luminesce through photochemical, chemical, and electrochemicalmeans. "Photoluminescence" is the process whereby a material is inducedto luminesce when it absorbs electromagnetic radiation. Fluorescence andphosphorescence are types of photoluminescence. "Chemiluminescent"processes entail the creation of luminescent species by chemicaltransfer of energy. "Electrochemiluminescence" entails creation ofluminescent species electrochemically.

Chemiluminescent assay techniques where a sample containing an analyteof interest is mixed with a reactant labeled with a chemiluminescentlabel have been developed. The reactive mixture is incubated and someportion of the labeled reactant binds to the analyte. After incubation,the bound and unbound fractions of the mixture are separated and theconcentration of the label in either or both fractions can be determinedby chemiluminescent techniques. The level of chemiluminescencedetermined in one or both fractions indicates the amount of analyte ofinterest in the biological sample.

Electrochemiluminescent (ECL) assay techniques are an improvement onchemiluminescent techniques. They provide a sensitive and precisemeasurement of the presence and concentration of an analyte of interest.In such techniques, the incubated sample is exposed to a voltammetricworking electrode in order to trigger luminescence. In the properchemical environment, such electrochemiluminescence is triggered by avoltage impressed on the working electrode at a particular time and in aparticular manner. The light produced by the label is measured andindicates the presence or quantity of the analyte. For a fullerdescription of such ECL techniques, reference is made to PCT publishedapplication US85/01,253 (W086/02734), PCT published application numberUS87/00,987, and PCT published application U.S. 88/03,947. Thedisclosures of the aforesaid applications are incorporated by reference.

It is desirable to carry out electrochemiluminescent assays without theneed for a separation step during the assay procedure and to maximizethe signal modulation at different concentrations of analyte so thatprecise and sensitive measurements can be made. Among prior art methodsfor nonseparation assays are those which employ microparticulate mattersuspended in the assay sample to bind one or more of the bindingcomponents of the assay.

U.S. Pat. No. 4,305,925 relates to the detection and determination ofclinically relevant proteins and peptides by means of nephelometric andturbidimetric methods. The methods disclosed involve binding the antigenor antibody to latex particles which perform the function of lightscattering or adsorption.

U.S. Pat. No. 4,480,042 relates to techniques employing particlereagents consisting of shell-core particles. The shell containsfunctional groups to which compounds of biological interest can becovalently bonded, and the high refractive index of the core results inhigh sensitivity to light scattering measurements. The technique isbased upon agglutination reactions which result from the reaction ofbivalent antibodies with multivalent antigens of interest to produceaggregates which can be detected and/or measured in various ways.

U.S. Pat. No. 4,419,453 likewise relates to the use of colored latexagglutination test methods useful for detecting the presence ofimmunochemicals such as antibodies and immunogens.

Based upon this prior art, it would not have appeared possible to usemicroparticulate matter in assays wherein a luminescent phenomenon ismeasured. One would expect that the luminescence from freechemiluminescent or electrochemiluminescent moieties would be absorbed,scattered, or otherwise suffer interference from the microparticulatematter.

Contrary to that expectation, U.S. application Ser. No. 539,389 (PCTpublished application U.S. 89/04,919) teaches sensitive, specificbinding assay methods based on a luminescent phenomenon wherein inertmicroparticulate matter is specifically bound to one of the bindingreactants of the assay system. The assays may be performed in aheterogeneous (one or more separation steps) assay format and may beused most advantageously in a homogeneous (nonseparation) assay format.

U.S. 89/04,919 relates to a composition for an assay based upon abinding reaction for the measurement of luminescent phenomenon, whichcomposition includes a plurality of suspended particles having a surfacecapable of binding to a component of the assay mixture. In anotheraspect, it is directed to a system for detecting or quantitating ananalyte of interest in a sample, which system is capable of conductingthe assay methods using the assay compositions of the inventions. Thesystem includes means for inducing the label compound in the assaymedium to luminesce, and means for measuring the luminescence to detectthe presence of the analyte of interest in the sample.

It was found that the binding of that component of the assay system towhich an electrochemiluminescent moiety has been linked, to suspendedmicroparticulate matter, greatly modulates the intensity of theluminescent signal generated by the electrochemiluminescent moietylinked to that component, thereby providing a means of monitoring thespecific binding reaction of the assay system. Even more surprisingly,the suspended particles were found to have little or no effect on theintensity of the luminescent signal generated by theelectrochemiluminescent moiety linked to the component of the systemwhich remains unbound to the suspended microparticulate matter.

Thus, U.S. 89/04,919 is directed to methods for the detection of ananalyte of interest in a sample, which method includes the steps of (1)forming a composition comprising (a) a sample suspected of containing ananalyte of interest, (b) an assay-performance-substance selected fromthe group consisting of (i) analyte of interest or analog of the analyteof interest, (ii) a binding partner of the analyte of interest or itssaid analog, and (iii) a reactive component capable of binding with (i)or (ii), wherein one of said substances is linked to a label compoundhaving a chemical moiety capable of being induced to luminesce, and (c)a plurality of suspended particles capable of specifically binding withthe analyte and/or a substance defined in (b) (i), (ii), or (iii); (2)incubating the composition to form a complex which includes a particleand said label compound; (3) inducing the label compound to luminesce;and (4) measuring the luminescence emitted by the composition to detectthe presence of the analyte of interest in the sample. Those samemethods may be used to quantify the amount of analyte in a sample bycomparing the luminescence of the assay composition to the luminescenceof a composition containing a known amount of analyte.

Analogs of the analyte of interest, which may be natural or synthetic,are compounds which have binding properties comparable to the analyte,but include compounds of higher or lower binding capability as well.Binding partners suitable for use in the present invention arewell-known. Examples are antibodies, enzymes, nucleic acids, lectins,cofactors and receptors. The reactive components capable of binding withthe analyte or its analog and/or with a binding partner thereof may be asecond antibody or a protein such as Protein A or Protein G or may beavidin or biotin or another component known in the art to enter intobinding reactions.

Advantageously, the luminescence arises from electrochemiluminescence(ECL) induced by exposing the label compound, whether bound or unboundto specific binding partners, to a voltammetric working electrode. TheECL reactive mixture is controllably triggered to emit light by avoltage impressed on the working electrode at a particular time and in aparticular manner to generate light. Although the emission of visiblelight is an advantageous feature the composition or system may emitother types of electromagnetic radiation, such as infrared orultraviolet light, X-rays, microwaves, etc. Use of the terms"electrochemiluminescence," "electrochemiluminescent""electrochemiluminescence" "luminescence," "luminescent," and"luminesce" includes the emission of light and other forms ofelectromagnetic radiation.

The methods taught in U.S. 89/04,919 permit the detection andquantitation of extremely small quantities of analytes in a variety ofassays performed in research and clinical settings. The demands ofresearchers and clinicians makes it imperative, however, to lower thedetection limits of assays performed by these methods to increase thesensitivities of those assays and to increase the speed at which theycan be performed.

Various methods are known in the art for increasing the signal fromlabeled species by concentrating them before subjecting them to ameasurement step. In U.S. Pat. No. 4,652,333, for example, particleslabeled with fluorescent, phosphorescent or atomic fluorescent labelsare concentrated by microfiltration before a measurement step isperformed.

It is also known in the art to concentrate labeled immunochemicalspecies prior to a measurement step, by, e.g., drawing magneticallyresponsive labeled particles to the surface of a measurement vessel. InU.S. Pat. Nos. 4,731,337, 4,777,145, and 4,115,535, for example, suchparticles are drawn to the vessel wall and then are irradiated to excitea fluorophoric emission of light.

In U.S. Pat. No. 4,945,045, particles are concentrated on a magneticelectrode. An electrochemical reaction takes place at the electrodefacilitated by a labeled chemical mediator. The immunochemical bindingreaction alters the efficiency of the mediator resulting in a modulatedsignal when binding takes place.

These prior art methods are not relevant to the surface selectiveexcitation processes of the invention. While not being bound by anyparticular mechanistic explanation of surface excitation, e.g.,electrochemiluminescence, it is believed that the label on thesolid-phase complex must be oxidized at the electrode. This requiresthat an electron move from the label to the electrode. It is believedthat the electron makes this "jump" by a phenomenon known as tunnelingin which the electron passes through space (a region where its potentialenergy is very high, e.g., the solution) without having to go "over" thepotential energy barrier. It can tunnel through the energy barrier, andthus, move from one molecule to another or from one molecule to anelectrode without additional energy input. However, this tunnelingphenomenon can only operate for very short distances. The probability ofthe tunneling phenomenon falls off exponentially as the distance betweenthe two species increases. The probability of the tunneling phenomenonoccurring between two species is fairly high if the distance is lessthan 25 Angstroms (2.5 nm) but is fairly low if the distance is greater.The distance of 25 Å is a rule-of-thumb used by those skilled in the artbut is not an absolute limitation.

Accordingly, only those ECL labels with 25 Å of the surface of theelectrode can be expected to participate in the ECL process. The area ofthe particle which is within 25 Å of the surface of an electrode istypically extremely small.

Accordingly, one would not expect that ECL from a particle surface wouldbe measurable to any significant degree. Moreover, the light which isproduced by the ECL process must pass through the particle to get to thephotomultiplier. Since the particles are essentially opaque (aconcentrated suspension of them is black) one would not expect that,even if significant amounts of light could be produced by ECL, that thelight could pass through the particle and be measured by thephotomultiplier.

OBJECTS OF THE INVENTION

It is therefore a primary object of this invention to providehomogeneous (non-separation) and heterogeneous (separation) methods,reagents and apparatus, for the conduct of binding assays.

It is a further object of this invention to provide non-separation,specific bonding assays, reagents and apparatus, based upon themeasurement of electrochemiluminescence emitted from an assaycomposition containing microparticulate matter.

It is a further and related object to provide such assays, reagents andapparatus having improved sensitivity, faster assay time, greatersensitivity, lower detection limits and greater precision than hasheretofore been achieved.

DESCRIPTION OF THE INVENTION DEFINITION OF TERMS

The term "ECL moiety," "metal-containing ECL moiety" "label," "labelcompound," and "label substance," are used interchangeably. It is withinthe scope of the invention for the species termed "ECL moiety,""metal-containing ECL moiety," "organometallic," "metal chelate,""transition metal chelate" "rare earth metal chelate," "label compound,""label substance" and "label" to be linked to molecules such as ananalyte or an analog thereof, a binding partner of the analyte or ananalog thereof, and further binding partners of such aforementionedbinding partner, or a reactive component capable of binding with theanalyte, an analog thereof or a binding partner as mentioned above. Theabove-mentioned species can also be linked to a combination of one ormore binding partners and/or one or more reactive components.Additionally, the aforementioned species can also be linked to ananalyte or its analog bound to a binding partner, a reactive component,or a combination of one or more binding partners and/or one or morereactive components. It is also within the scope of the invention for aplurality of the aforementioned species to be bound directly, or throughother molecules as discussed above, to an analyte or its analog. Forpurposes of brevity, these ligands are referred to as anassay-performance-substance.

The terms detection and quantitation are referred to as "measurement",it being understood that quantitation may require preparation ofreference compositions and calibrations.

The terms collection and concentration of complex may be usedinterchangeably to describe the concentration of complex within theassay composition and the collection of complex at, e.g., an electrodesurface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a cell for performing themicroparticulate-based nonseparation and separation assays of theinvention.

FIG. 2 is a simplified diagram of a voltage control apparatus for usewith the cell of FIG. 1.

FIG. 3 is a schematic representation of a direct incorporation PCRformat using electrochemiluminescent labeled oligonucleotides and biotinelectrochemiluminescent labeled oligonucleotides as primers.

FIG. 4 is a schematic representation of a normal PCR format using abiotinylated primer to allow the generation of biotinylated PCR andPRODUCT.

FIG. 5 is a schematic representation of an asymmetric PRC assay formatgenerating single-stranded biotinylated DNA for later hybridization toelectrochemiluminescent labeled oligonucleotides.

FIG. 6 is a graph showing specificity studies of the directincorporation of electrochemiluminescent labeled oligonucleotides intobiotinylated PCR products.

FIG. 7 is a standard curve of directly incorporatedelectrochemiluminescent label and biotinylated oligonucleotides intoHPV16 PCR products.

FIG. 8 is a graph showing a point mutation assay for the Ha-rasoncogene.

FIG. 9 is a graph showing an evaluation of the specificity ofelectrochemiluminescent labeled probes using P³² electrochemiluminescentlabeled probes for the Aa-ras oncogene.

FIG. 10 is a graph showing the determination of the relative value ofelectrochemiluminescent label in P³² electrochemiluminescent label forthe determination of point mutations in the Ha-ras oncogene.

FIG. 11 is a standard curve of the rapid "no wash" hybridization assayfor HPV18.

FIG. 12 is a schematic representation of an assay cell used to conductassays relying upon gravitational force to cause the complex to settle.

FIG. 13 is a graph showing the distance the complex settles as afunction of time under influence of gravity.

FIG. 14 is a graph showing the intensity of electrochemiluminescence asa function of time in gravity cells having different heights of assaycomposition over the electrode.

FIG. 15 is a graph showing the intensity of electrochemiluminescence ingravity cells having different heights of assay composition over theelectrode surface as measured in an assay for the measurement of alphafetal protein.

FIG. 16 is a schematic representation of a sedimentation assay cellwhich employs an electromagnet to cause the complex to settle on theelectrode surface.

FIG. 17 is a graph showing the relative rates of settling ofmicroparticulate complex under the influence of a magnetic field and ofgravity, respectively.

FIG. 18 is a schematic representation of a collection cell including apermanent magnet.

FIG. 19 is a graph showing the increase in ECL intensity as a functionof time in assays conducted with the cell of FIG. 18.

FIG. 20(A-B) is a schematic representation of the lines of force in thevicinity of the electrode surface as a function of the orientation ofthe magnet beneath the electrode surface.

FIG. 21 is a schematic representation of a rotary flow cell wherein thecomplexes are deposited upon the surface of the electrode bycentrifugation.

FIG. 22 is a schematic representation of an evanescent-wave fluorescencedetection.

Fig. 23 is a schematic representation of a flow cell equipped with amagnet system which imposes magnetic field lines largely parallel to theplane of the working electrode surface.

FIG. 24 is a schematic representation of a magnet system useful in theflow cell of FIG. 23.

BRIEF DESCRIPTION OF THE INVENTION

In its broadest embodiment, the invention is in a method for performinga binding assay for an analyte of interest present in a sample. Thesteps include:

(a) forming a composition containing

(i) said sample

(ii) an assay-performance-substance which contains a component linked toa label compound capable of being induced to luminesce, and

(iii) a plurality of particles capable of specifically binding with theanalyte and/or said assay-performance-substance;

(b) incubating said composition to form a complex which includes aparticle and said label compound;

(c) collecting said complex in a measurement zone;

(d) inducing the label compound in said complex to luminesce by surfaceselective excitation; and

(e) measuring the emitted luminescence to measure the presence of theanalyte of interest in the sample.

The complex may be collected on, e.g., an electrode surface where it isexcited and induced to electrochemiluminesce, as by impressing a voltageon the electrode, or, it may be collected on a surface and be thereafterinduced to fluoresce by surface excitation as described below.Total-internal-reflection-fluorescence (TIRF) has been described citereference! as a surface sensitive technique for exciting and detectingfluorophoric labels and total-internal-reflection has been used withRAMAN cite reference! and infra-red absorption cite reference! asanother surface-sensitive technique for measuring the presence of alabel. Surface plasmon resonance is an optical technique which may beused according to methods of the invention to measure labels onsurfaces. The invention is thus directed to methods for excitingluminescence by surface excitation techniques.

While the invention is preferably carried out by collecting the complexin a measurement zone, i.e., on a surface at which it can be caused toluminesce, the invention also embraces methods wherein the complex iscollected in a measurement zone and thereafter means are brought to thatzone or other steps taken to induce and measure luminescence.

The collection of the complex may be carried out by several differentmethods, including gravity settling, filtration, centrifugation andmagnetic attraction of magnetically responsive particles which form partof the complex. The several embodiments are described in further detailbelow.

Assays based upon the measurement of electrochemiluminescence at anelectrode surface are advantageously carried out using the forces ofgravity by

(a) forming a composition containing

(i) said sample

(ii) an assay-performance-substance which contains a component linked toa label compound capable of being induced to electrochemiluminesce, and

(iii) a plurality of suspended particles having a density greater thanthe balance of said composition and being capable of specificallybinding with the analyte and or said assay-performance-substance;

(b) incubating said composition to form a complex which includes aparticle and said label compound;

(c) introducing said composition into an assay cell;

(d) collecting said complex at the surface of an electrode located belowat least a substantial portion of the volume of said assay cell bypermitting said composition to reside in said cell for a time sufficientto permit the particles to settle upon said electrode surface by theforce of gravity;

(e) inducing the label compound in said collected complex toluminescence by imposing a voltage on said electrode; and

(f) measuring the emitted luminescence at the electrode surface tomeasure the presence of the analyte of interest in the sample.

While batch assays can be performed, continuous or semi-continuousassays can be performed in flow cells. In a flow cell, the solid-phaseremains in the measurement celthrhile the solution flows through andexits the cell. If the solid-phase (e.g., particles) are more dense thanwater, i.e., have a density greater than that of water, (more than 1.0g/mL) the force of gravity upon the particles causes them to fall to thebottom of the cell. The cell can be constructed such that the particlessettle to the bottom as the fluid flows through the cell or the cell canbe constructed such that the majority of the sample is contained in thecell in a columnar compartment above the working electrode of an ECLsystem. Sufficient dwell time in the cell must be provided to permit theparticles to settle on the surface of the electrode before inducing ECL.

In another embodiment of the invention, the assay composition containingsuspended particles having a density greater than the balance of theassay composition may be subjected to centrifugation in order to removethe particles to a measurement zone where they are subsequently broughtinto contact with, e.g., an electrode to induce electrochemiluminescenceor brought directly into contact with an electrode in the centrifugationstep.

In this embodiment, the measurement cell is provided with means torapidly rotate the sample and sample enclosure. Centrifugal force causesthe particles in the sample to move outward from the axis of rotation ofthe sample enclosure and to collect on the outer surface of the sampleenclosure. The outer surfaces of such sample enclosure may constitutethe working electrode of an ECL measurement system.

In a third embodiment, the particles may be removed by filtration fromthe assay composition. In this embodiment the particles need not have adensity greater than the balance of the assay composition. Theinvention, the particles are separated from the solution andconcentrated by drawing the solution through a filter, e.g. pumping andcollecting the particles on the surface of the filter. This surface ofthe filter is, for example, coated with a thin metal film which canserve as the working electrode in an ECL detection system.

In a preferred embodiment, the suspended particles are magneticallyresponsive, e.g. they may be paramagnetic or ferromagnetic, and arecollected in a measurement zone or, preferably, directly at the surfaceof an electrode, by imposition of a magnetic field on the particles. Themeasurement cell is equipped with a magnet. The magnetic field of themagnet applies a force on the particles as they reside in a batch cellor as they flow through a flow cell, causing them to separate from thebulk of the solution onto the surface of the cell which is in closestproximity to the magnet. If the magnet is placed in a proper orientationand in close proximity to the working electrode of an ECL detectionsystem the particles will concentrate on the surface of the workingelectrode.

Several different heterogeneous and homogeneous formats for bindingassays can be implemented using the methods described above to collectand concentrate the complex on the surface of an electrode. In aheterogeneous binding assay the complex is separated from thecomposition before measuring luminescence from the label. In homogeneousassays, no separation of the bound (to the solid phase) and unboundlabeled reagents is made.

In a homogeneous assay, when the complex is concentrated on the surfaceof the working electrode, the measured signal from the label is muchgreater than it would be in the absence of a collection step. The signalfrom the uncomplexed labeled reagents, in contrast, is not changed.Hence, despite the presence of the uncomplexed labeled reagents in themeasurement cell, the signal from the collected complex is stronger thanin an assay without collection of complex. The detection limit for thebinding assay is, much improved as a result of the collection procedure.

In a preferred embodiment of the invention, an in-situ separation stepis included in the homogeneous binding assay procedure. After the assaycomposition, i.e., sample, assay performance substance and particleshave been pumped into the measurement cell and the complex captured uponthe working electrode, a second fluid is pumped through the cell whichis free of label or labeled reagents, thereby performing an in-situ washor separation of the complex from unbound components of the assaycomposition. This assay procedure is technically a heterogeneous bindingassay. However, the ability to perform the separation inside themeasurement cell is advantageous in that it does not require additionalseparation apparatus and the procedure is generally much faster thanexternal separation methods.

Heterogeneous binding assays are conducted using the invention by mixingthe components of the assay composition and allowing them to react for apredetermined length of time. The assay composition is then subjected toa separation step wherein the solution is separated from the particles.Electrochemiluminescence is then measured from either the complex or thesolution. Measuring the ECL from the complex after a concentration steppermits measurement of analyte with better accuracy and with a lowerdetection limit than is possible without concentration.

DETAILED DESCRIPTION OF THE INVENTION

The invention, as well as other objects and features thereof, will beunderstood more clearly and fully from the following description ofcertain preferred embodiments.

The invention is broadly applicable to analytes of interest which arecapable of entering into binding reactions. These reactions include,e.g., antigen-antibody, ligand receptor, DNA and RNA interactions, andother known reactions. The invention relates to different methods andassays for qualitatively and quantitatively detecting the presence ofsuch analytes of interest in a multicomponent sample.

The Samples

The sample which may contain the analyte of interest, which may be insolid, emulsion, suspension, liquid, or gas form, may be derived from,for example, cells and cell-derived products, water, food, blood, serum,hair, sweat, urine, feces, tissue, saliva, oils, organic solvents orair. The sample may further comprise, for example, water, acetonitrile,dimethyl sulfoxide, dimethyl formamide, n-methyl-pyrrolidone or alcoholsor mixtures thereof.

The Analytes

Typical analytes of interest are a whole cell or surface antigen,subcellular particle, virus, prion, viroid, antibody, antigen, hapten,fatty acid, nucleic acid, protein, lipoprotein, polysaccharide,lipopolysaccharide, glycoprotein, peptide, polypeptide, cellularmetabolite, hormone, pharmacological agent, synthetic organic molecule,organometallic molecule, tranquilizer, barbiturate, alkaloid, steroid,vitamin, amino acid, sugar, lectin, recombinant or derived protein,biotin, avidin, streptavidin, or inorganic molecule present in thesample. Typically, the analyte of interest is present at a concentrationof 10⁻³ molar or less, for example, as low as 10⁻¹² molar or lower.

Assay-Performance-Substance

The assay-performance-substance which is combined with the samplecontaining the analyte of interest contains at least one substanceselected from the group consisting of (i) added analyte of interest orits analog, as defined above, (ii) a binding partner of the analyte ofinterest or its said analog, and (iii) a reactive component, as definedabove, capable of binding with (i) or (ii), wherein one of saidsubstances is linked to a compound or moiety, e.g. an ECL moiety capableof being induced to luminesce. The labeled substance may be a whole cellor surface antigen, a subcellular particle, virus, prion, viroid,antibody, antigen, hapten, lipid, fatty acid, nucleic acid,polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein,peptide, polypeptide, cellular metabolite, hormone, pharmacologicalagent, tranquilizer, barbiturate, alkaloid, steroid, vitamin, aminoacid, sugar, nonbiological polymer (preferably soluble), lectin,recombinant or derived protein, synthetic organic molecule,organometallic molecule, inorganic molecule, biotin, avidin orstreptavidin. In one embodiment, the reagent is anelectrochemiluminescent moiety conjugated to an antibody, antigen,nucleic acid, hapten, small nucleotide sequence, oligomer, ligand,enzyme, or biotin, avidin, streptavidin, Protein A, Protein G, orcomplexes thereof, or other secondary binding partner capable of bindingto a primary binding partner through protein interactions.

Analogs of the analyte of interest, which can be natural or synthetic,are typically compounds which have binding properties comparable to theanalyte, but can also be compounds of higher or lower bindingcapability. The reactive components capable of binding with the analyteor its analog, and/or with a binding partner thereof, and through whichthe ECL moiety can be linked to the analyte, is suitably a secondantibody or a protein such as Protein A or Protein G, or avidin orbiotin or another component known in the art to enter into bindingreactions.

The Labels

Advantageously, the ECL moieties are metal chelates. The metal of thatchelate is suitably any metal such that the metal chelate will luminesceunder the electrochemical conditions which are imposed on the reactionsystem in question. The metal of such metal chelates is, for instance, atransition metal (such as a d-block transition metal) or a rare earthmetal. The metal is preferably ruthenium, osmium, rhenium, iridium,rhodium, platinum, indium, palladium, molybdenum, technetium, copper,chromium or tungsten. Especially preferred are ruthenium and osmium.

The ligands which are linked to the metal in such chelates are usuallyheterocyclic or organic in nature, and play a role in determiningwhether or not the metal chelate is soluble in an aqueous environment orin an organic or other nonaqueous environment. The ligands can bepolydentate, and can be substituted. Polydentate ligands includearomatic and aliphatic ligands. Suitable aromatic polydentate ligandsinclude aromatic heterocyclic ligands. Preferred aromatic heterocyclicligands are nitrogen-containing, such as, for example, bipyridyl,bipyrazyl, terpyridyl, and phenanthrolyl. Suitable substituents includefor example, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl,substituted aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano,amino, hydroxy, imino, hydroxycarbonyl, aminocarbonyl, amidine,guanidinium, ureide, sulfur-containing groups, phosphorus containinggroups, and the carboxylate ester of N-hydroxysuccinimide. The chelatemay have one or more monodentate ligands, a wide variety of which areknown to the art. Suitable monodentate ligands include, for example,carbon monoxide, cyanides, isocyanides, halides, and aliphatic, aromaticand heterocyclic phosphines, amines, stilbenes, and arsines.

Examples of suitable chelates are bis(4,4'-carbomethoxy)-2,2'-bipyridine!2-3-(4-methyl-2,2'-bipyridine-4-yl)propyl!-1,3-dioxolane ruthenium (II);bis(2,2'bipyridine) 4-(butan-1-al)-4'-methyl-2,2'-bipyridine!ruthenium(II); bis(2,2'-bipyridine) 4-(4'-methyl-2,2'-bipyridine-4'-yl)-butyricacid!ruthenium (II); tris(2,2'bipyridine)ruthenium (II);(2,2'-bipyridine) bis-bis(1,2-diphenylphosphino)ethylene!2-3-(4-methyl-2,2'-bipyridine-4'-yl)propyl!-1,3-dioxolane osmium (II);bis(2,2'-bipyridine) 4-(4'-methyl-2,2'-bipyridine)-butylamine!ruthenium(II); bis(2,2'-bipyridine)1-bromo-4(4'-methyl-2,2'-bipyridine-4-yl)butane!ruthenium (II);bis(2,2'-bipyridine)maleimidohexanoic acid,4-methyl-2,2'-bipyridine-4'-butylamide ruthenium (II). Other ECLmoieties are described in commonly assigned PCT published applicationUS87/00,987 and PCT published application 88/0394.

The function of the ECL moieties is to emit electromagnetic radiation asa result of introduction into the reaction system of electrochemicalenergy. In order to do this, they must be capable of being stimulated toan excited energy state and also capable of emitting electromagneticradiation, such as a photon of light, upon descending from that excitedstate. While not wishing to be bound by theoretical analysis of themechanism of the ECL moiety's participation in theelectrochemiluminescent reaction, we believe that it is oxidized by theintroduction of electrochemical energy into the reaction system andthen, through interaction with a reductant present in the system, isconverted to the excited state. This state is relatively unstable, andthe metal chelate quickly descends to a more stable state. In so doing,the chelate gives off electromagnetic radiation, such as a photon oflight, which is detectable.

The amount of metal chelate or other metal-containing ECL moietyincorporated in accordance with the invention will vary from system tosystem. Generally, the amount of such moiety utilized is that amountwhich is effective to result in the emission of a detectable, and ifdesired, quantitatable, emission of electromagnetic energy, from theaforementioned composition or system. The detection and/or quantitationof an analyte of interest is typically made from a comparison of theluminescence from a sample containing an analyte of interest and an ECLmoiety to the luminescence emitted by a calibration standard developedwith known amounts of the analyte of interest and ECL moiety. Thisassumes a homogeneous format. In the heterogeneous mode, a separation asdiscussed previously is carried out prior to ECL analysis.

As can be appreciated by one of ordinary skill in the art, the identityand amount of the metal-containing ECL moiety will vary from one systemto another, depending upon prevailing conditions. The appropriatemetal-containing ECL moiety, and sufficient amount thereof to obtain thedesired result, can be determined empirically by those of ordinary skillin the art, once equipped with the teachings herein, without undueexperimentation.

The Particles

The particles advantageously comprise micro-particulate matter having adiameter of 0.05 um to 200 um, preferably 0.1 um to 100 um, mostpreferably 0.5 um to 10 um, and a surface component capable of bindingto the analyte and/or one or more of the other substances defined insubparagraphs (b) (i), (b) (ii), or (b) (iii) above. For example, themicroparticulate matter may be crosslinked starch, dextrans, cellulose,proteins, organic polymers, styrene copolymer such as styrene/butadienecopolymer, acrylonitrile/butadiene/styrene copolymer, vinylacetylacrylate copolymer, or vinyl chloride/acrylate copolymer, inertinorganic particles, chromium dioxide, oxides of iron, silica, silicamixtures, and proteinaceous matter, or mixtures thereof. Desirably, theparticles are suspended in the ECL system.

Assay Media

In order to operate a system in which an electrode introduceselectrochemical energy, it is necessary to provide an electrolyte inwhich the electrode is immersed and which contains the ECL moiety. Theelectrolyte is a phase through which charge is carried by ions.Generally, the electrolyte is in the liquid phase, and is a solution ofone or more salts or other species in water, an organic liquid ormixture of organic liquids, or a mixture of water and one or moreorganic liquids. However, other forms of electrolyte are also useful incertain embodiments of the invention. For example, the electrolyte maybe a dispersion of one or more substances in a fluid--e.g., a liquid, avapor, or a supercritical fluid--or may be a solution of one or moresubstances in a solid, a vapor or supercritical fluid.

The electrolyte is suitably a solution of a salt in water. The salt canbe a sodium salt or a potassium salt preferably, but incorporation ofother cations is also suitable in certain embodiments, as long as thecation does not interfere with the electrochemiluminescent interactionsequence. The salt's anion may be a phosphate, for example, but the useof other anions is also permissible in certain embodiments of theinvention--once again, as long as the selected anion does not interferewith the electrochemiluminescent interaction sequence.

The composition may also be nonaqueous. While supercritical fluids canin certain instances be employed advantageously, it is more typical toutilize an electrolyte comprising an organic liquid in a nonaqueouscomposition. Like an aqueous electrolyte, the nonaqueous electrolyte isalso a phase through which charge is carried by ions. Normally, thismeans that a salt is dissolved in the organic liquid medium. Examples ofsuitable organic liquids are acetonitrile, dimethylsulfoxide (DMSO),dimethylformamide (DMF), methanol, ethanol, and mixtures of two or moreof the foregoing. Illustratively, tetraalkylammonium salts, such astetrabutylammonium tetrafluoroborate, which are soluble in organicliquids can be used with them to form nonaqueous electrolytes.

The electrolyte is, in certain embodiments of the invention, a bufferedsystem. Phosphate buffers are often advantageous. Examples are anaqueous solution of sodium phosphate/sodium chloride, and an aqueoussolution of sodium phosphate/sodium fluoride.

Other Assay Components

As described PCT published application U.S. 89/04859 commonly assigned,entitled Electrochemiluminescent Reaction Utilizing Amine-DerivedReductant, the disclosure of which is incorporated by reference, it isdesirable to include a reductant, typically an amine or amine moiety (ofa larger molecule) which can be oxidized and spontaneously decomposed toconvert it into a highly reducing species. It is believed that the amineor amine moiety is also oxidized by electrochemical energy introducedinto the reaction system. The amine or amine moiety loses one electron,and then deprotonates, or rearranges itself, into a strong reducingagent. This agent interacts with the oxidized metal-containing ECLmoiety and causes it to assume the excited state discussed above. Inorder to carry out its role, the amine or amine moiety preferably has acarbon-centered radical with an electron which can be donated from suchcarbon, and an alpha carbon which can then act as a proton donor duringdeprotonation in order to form the reductant. The amine-derivedreductant provides the necessary stimulus for converting themetal-containing ECL moiety to its excited state, from which detectableelectromagnetic radiation is emitted.

A wide range of amines and corresponding amine moieties can be utilizedin practicing the present invention. Generally, the amine or aminemoiety is chosen to suit the pH of the system which is to beelectrochemiluminescently analyzed. Another relevant factor is that theamine or amine moiety should be compatible with the environment in whichit must function during analysis, i.e., compatible with an aqueous ornonaqueous environment, as the case may be. Yet another consideration isthat the amine or amine moiety selected should form an amine-derivedreductant under prevailing conditions which is strong enough to reducethe oxidized metal-containing ECL moiety in the system.

Amines (and corresponding moieties derived therefrom) which areadvantageously utilized in the present invention are aliphatic amines,such as primary, secondary and tertiary alkyl amines, the alkyl groupsof each having from one to three carbon atoms, as well as substitutedaliphatic amines. Tripropyl amine is an especially preferred amine as itleads to, comparatively speaking, a particularly high-intensity emissionof electromagnetic radiation, which enhances the sensitivity andaccuracy of detection and quantitation with embodiments in which it isused. Also suitable are diamines, such as hydrazine, and polymines, suchas poly(ethyleneimine). Examples of other amines suitable for practicingthe invention are triethanol amine, triethyl amine,1,4-diazabicyclo(2.2.2)-octane, 1-piperidine ethanol,1,4-piperazinebis-(ethane-sulfonic acid), tri-ispropyl amine andpoly(ethyleneimine).

Typically, the metal-containing ECL moiety utilized in the presentinvention is the reaction-limiting constituent. Accordingly, it is alsotypical that the amine or amine moiety is provided in a stoichiometricexcess with respect thereto. Illustratively, the amine or amine moietyis employed in a concentration of 50-150 mM. For utilization at a pH ofapproximately 7, a concentration of 100 mM is often advantageous. Incertain embodiments, the upper limit on amine or amine moietyconcentration is determined by the maximum solubility of the amine ormoiety in the environment in which it is being used, for example inwater. In general, the amount of amine or amine moiety employed is thatwhich is sufficient to effect the transformation of the oxidizedmetal-containing ECL moiety into its excited state so that luminescenceoccurs. Those of ordinary skill in the art, equipped with the teachingsherein, can determine empirically the amount of amine or amine moietyadvantageously used for the particular system being analyzed, withoutundue experimentation.

As described in commonly assigned PCT published application US 89/04915,entitled Enhanced Electrochemiluminescent Reaction, the contents ofwhich are incorporated by reference, the assays of the invention aredesirably carried out in the presence of an enhancer, typically acompound of the formula

    R-- --(OR').sub.x OH

wherein R is hydrogen or C_(n) H_(n2+1), R' is C_(n) H_(2n), x is 0 to70, and n is from 1 to 20. Specifically, n is from 1 to 4. Specificexamples are a substance available in commerce under the name TritonX-100, of the formula ##STR1## wherein x is 9-10, and a substanceavailable in commerce under the name Triton N-401 (NPE-40), of theformula

    C.sub.9 H.sub.19 -- --(OCH.sub.2 CH.sub.2).sub.x --OH

wherein x is 40. The enhancer is generally utilized in an amountsufficient so that in its presence the desired increase in emission ofelectromagnetic radiation occurs. Typically, the amount is 0.01% to5.0%, more specifically 0.1% to 1.0%, v/v. The subject matter of thisapplication is incorporated by reference.

The ECL moiety incorporated in accordance with the present invention isinduced to emit electromagnetic radiation by stimulating it into anexcited state. This is accomplished by exposing the system in which theECL moiety is incorporated to electrochemical energy. The potential atwhich oxidation of the ECL moiety and the species forming a strongreductant occurs depends upon the exact chemical structures thereof, aswell as factors such as the pH of the system and the nature of theelectrode used to introduce electrochemical energy. It is well known tothose of ordinary skill in the art how to determine the optimalpotential and emission wavelength of an electrochemiluminescent system.Certain preferred methods of stimulating the ECL system are disclosed incommonly assigned PCT published application US 89/01,814, the contentsof which are incorporated herein by reference.

Apparatus for Measuring Electrochemiluminescence

An apparatus for carrying out the assays of the invention is describedin FIGS. 1 and 2. FIG. 1 discloses an advantageous ECL apparatus, butthe methods of the present invention are not limited to application inapparatus 10, but rather may be employed in other types of ECL apparatuswhich include a working electrode or other triggering surface to provideelectrochemical energy to trigger the ECL moiety intoelectrochemiluminescence. While the methods of the invention can becarried out in a static or flow-through mode, apparatus 10 includes aflow-through cell, which provides distinct advantages for many types ofsamples including binding assay samples. Further details of apparatusfor carrying out the ECL assays of the invention are disclosed incommonly assigned published PCT applications US 89/04,854 and U.S.90/01,370.

Apparatus 10 includes an electrochemical cell 12, a lightdetection/measurement device 14, which may advantageously be aphotomultiplier tube (PMT), photodiode, charge coupled device,photographic film or emulsion or the like, and a pump 16, which isadvantageously a peristaltic pump, to provide for fluid transport to,through and from cell 12. Alternatively, a positive displacement pumpmay be used. A shutter mechanism 18 is provided between cell 12 and PMT14 and is controllably operated to open only so far as to expose PMT 14to cell 12 during ECL measurement periods. The shutter mechanism may beclosed, for example, during maintenance. Also included in apparatus 10but not illustrated in FIG. 1 is a lightproof housing intended to mountthe various components therein and to shield PMT 14 from any externallight during the ECL measurements.

Cell 12 itself includes a first mounting block 20 through which passesan inlet tube 22 and an outlet tube 24, which may be advantageouslyconstructed of stainless steel. Mounting block 20 has a first, outersurface 26 and a second, inner surface 28 defining one side of asample-holding volume 30 of cell 12 in which cell 12 holds the cleaningand/or conditioning and/or measurement solutions during correspondingoperations of apparatus 10. Inlet and outlet tubes 22, 24 pass throughmounting block 20 from outer surface 26 to inner surface 28 and openinto sample-holding volume 30. A second mounting block 32,advantageously constructed of stainless steel also has a first, outersurface 34 and a second, inner surface 36. Second mounting block 32 isseparated from first mounting block 20 by an annular spacer 38,advantageously constructed of Teflon or other non-contaminable material.Thus, outer surface 34 of mounting block 30 defines part of the secondside of the sample-holding volume 30. Spacer 38 has an outer portion 40and a central aperture 42 whose inner edge 44 defines the side wall ofsample-holding volume 30. Outer portion 40 seals the inner surface 28 offirst mounting block 20 to outer surface 34 of second mounting block 32to prevent any solution from passing out from sample-holding volume 30between the two surfaces 28, 34. Mounting block 32 further has a centralaperture 46 in which a window 48 is seal-fitted to define the rest ofthe second side of sample-holding volume 30 as a continuation of outersurface 34. Window 48 is formed of a material which is substantiallytransparent at the wavelength of electrochemiluminescent light emittedby the ECL moiety. Window 48 is therefore advantageously formed ofglass, plastic, quartz or the like.

Inlet tube 22 intersects sample-holding volume 30 at a first end 50thereof adjacent to spacer 38 and outlet tube 24 intersectssample-holding volume 30 at a second end 52 thereof, adjacent spacer 38.The combination of inlet tube 22, sample-holding volume 30 and outlettube 24 thereby provides a continuous flow path for the narrow,substantially laminar flow of a solution to, through and from cell 12.

Mounted on inner surface 28 of first mounting block 20 is a workingelectrode system 54 which, in the illustrated embodiment, includes firstand second working electrodes 56 and 58. In other embodiments, a singleworking electrode may advantageously be provided, or only electrode 56may be a working electrode. Working electrodes 56, 58 are where theelectrochemical and ECL reactions of interest can take place. Workingelectrodes 56, 58 are solid voltammetric electrodes and may therefore beadvantageously constructed of platinum, gold, carbons or other materialswhich are effective for this purpose. Wire connectors 60, 62 connectedto working electrodes 56, 58, respectively, pass out through firstmounting block 20.

Connectors 60, 62 are both connected to a first, "working electrode"terminal 64 of a voltage control 66, illustrated in FIG. 2. Voltagecontrol 66 advantageously operates in the manner of a potentiostat tosupply voltage signals to working electrodes 56, 58 and optionally tomeasure current flowing therefrom during an ECL measurement.Alternatively, connectors 60, 62 may be connected to separate terminalsof voltage control 66 for individual operation.

The potentiostat operation of voltage control 66 is further effectedthrough a counter electrode 68 and, optionally but advantageously, areference electrode 70. In the illustrated embodiment, mounting block 32is made of stainless steel and counter electrode 68 consists in exposedsurfaces 72, 74 of mounting block 32. Counter electrode 72, 74 andworking electrodes 56, 58 provide the interface to impress the potentialon the solution within sample-holding volume 30 which energizes thechemical reactions and triggers electrochemiluminescence in the sampleand/or provides energy for cleaning and conditioning the surfaces ofcell 12. Counter electrode 72, 74 is connected by a wire connector 76 toa second, "counter electrode" terminal 78 of voltage control 66.

Reference electrode 70 provides a reference voltage to which the voltageapplied by the working electrodes 56, 58 is referred, for example, +1.2volts versus the reference. Reference electrode 70 is advantageouslylocated in outlet tube 24 at a position 80 spaced from cell 12 and isconnected through a wire connector 82 to a third "reference electrode"terminal 84 of voltage control 66. In the three electrode mode, currentdoes not flow through reference electrode 70. Reference electrode 70 maybe used in a three electrode mode of operation to provide a poised,known and stable voltage and is therefore advantageously constructed ofsilver/silver chloride (Ag/AgCl) or is a saturated calomel electrode(SCE). Voltage control 66 may be operable in a two electrode mode ofoperation using only working electrode 56 and electrode 58 as acounter/reference electrode. In this two electrode mode of operation,counter/reference electrode 58 is electrically connected to voltagecontrol terminals 78 and 84 on voltage control 66. In this case, voltagecontrol 66 operates essentially as a battery. Voltage control 66supplies voltage signals to working and counter electrodes 56 and 58 andoptionally measures the current flowing through the respectiveelectrodes. Reference electrode 70 may alternatively be a so-called"quasi-reference" electrode constructed of platinum, gold, stainlesssteel or other material, which provides a less stable voltage, yet onethat is measurable with respect to the solution in contact. In both thetwo and three electrode mode, the reference electrode 70 or 58 servesthe purpose of providing a reference against which the voltage appliedto working electrodes 56 is measured. The poised voltage reference iscurrently considered to be more advantageous. Voltage control 66 in itspotentiostat operation controls the various electrodes by providing aknown voltage at working electrodes 56, 58 with respect to referenceelectrode 70 while measuring the current flow between working electrodes56, 58 and counter electrode 72, 74. Potentiostats for this purpose arewell known, and the internal structure of voltage control 66 maytherefore correspond to any of the conventional, commercially availablepotentiostats which produce the above-recited functions functions and sodo not form a part of the present invention per se. Indeed, apparatus 10may alternatively be constructed without an internal voltage control 66,and may be adapted to be connected to an external potentiostat which isseparately controlled for providing the required voltage signals toelectrodes 56, 58, 72, 74 and 70. These voltage signals, applied in aspecific manner as described below, provide repeatable initialconditions for the surfaces of working electrodes 56, 58 andadvantageously for the surfaces of cell 12 as a whole, a feature whichcontributes significantly to improved precision in ECL measurements.

Pump 16 is advantageously positioned at outlet tube 24 to "pull"solution from a sample volume in the direction of arrow A into inlettube 22. The solution will flow through inlet tube 22, sample-holdingvolume 30 and outlet tube 24 past reference electrode 70 and out in thedirection of arrow B. Alternatively, pump 16 may be positioned at inlettube 22 to "push" the solution through apparatus 10. Advantageously,this same flow path through inlet tube 22, sample-holding volume 30 andoutlet tube 24 is used for all solutions and fluids which pass throughcell 12, whereby each fluid performs a hydrodynamic cleaning action inforcing the previous fluid out of cell 12. Pump 16 may be controlled tosuspend its operation to hold a particular solution in cell 12 for anyperiod of time.

The flow-through construction of apparatus 10 permits working electrodesto be impressed with a variable voltage or to be continuously held at apreoperative potential while being continuously exposed to one or moresolutions without exposing working electrodes 56, 58 (or counter andreference electrodes 72, 74, 70) to air. Exposure to air, which opensthe circuit to the reference electrode 70, permits unknown, randomvoltage fluctuations which destroy the reproducibility of surfaceconditions on working electrodes 56, 58. The flow-through constructionpermits the rapid alternation between initializing steps, in whichelectrode system 54 is cleaned and conditioned, and measurement steps,in which one or more measurement waveforms or sweeps trigger ECL.

The invention is also directed to reagent compositions. Broadly, thereagent compositions may be any one of the components of the assaysystems of the invention, i.e., (a) electrolyte, (b) label compoundcontaining an ECL moiety, (c) particles, including magneticallyresponsive particles, (d) analyte of interest or an analog of theanalyte of interest, (e) a binding partner of the analyte of interest orof its analog, (f) a reactive component capable of reacting with (d) or(e), (g) a reductant, or (h) an electrochemiluminescence-reactionenhancer. The reagents may be combined with one another for convenienceof use, i.e., two component, three component, and higher multiplecomponent mixtures may be prepared, provided that the components are notreactive with one another during storage so as to impair their functionin the intended assay. Desirably, the reagents are two-component ormulticomponent mixtures which contain particles as well as one or moreother components.

The invention is also directed to kits. The kits may include vesselscontaining one or more of the components (a) to (h) recited above or thekits may contain vessels containing one or more reagent compositions asdescribed above comprising mixtures of those components, all for use inthe assay methods and systems of the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

While a wide range of particles can be employed in the particle-basedassays of the invention, generally the particles have a density of from1.0 to 5.0 g/mL and preferably have a density of from 1.1 to 2 g/mL.Choice of the optimum density is within the skill of the art, the rateof settling in gravity-driven assays being a trade-off between the speedof the assay and the desire to create a uniform layer of complex on theelectrode surface.

Particles having a wide range of mean diameters can also be employed.Particles having a mean diameter of from 0.001 to 100 μm can be used andpreferably the particles have a mean diameter of from 0.01 to 10 μm.

Wide ranges of concentration of particles in the assay composition canalso be employed. For example, the concentration can range from 1 to10,000 μg/mL to preferably from 5 to 1000 μg/mL. Desirably, the densityof the particles, their size and their concentration is selected suchthat the particles settle at a rate of at least 0.5 mm/min andpreferably at a faster rate.

In the filtration mode of performing the invention, the filtration meansdesirably has a pore size, measured as mean diameter, from broadly 0.01to 90% of the mean diameter of the particles and preferably from 10% to90% of that diameter.

The art has described a number of magnetic particles which can be usedin the assays of the invention. For example, U.S. Pat. Nos. 4,628,037,4,695,392, 4,695,393, 4,698,302, 4,554,088, U.K. Patent Application GB2,005,019A and EP 0,180,384, describe a variety of magnetic particleswhich can be used with success. The particles may be paramagnetic orferromagnetic and may be coated with various materials to which bindingcompounds are coupled so that the magnetic particle can be used inimmunoassays. Desirably the magnetic particles used in the inventionhave a susceptibility of at least 0.001 cgs units and desirably thesusceptibility is at least 0.01 cgs units. The magnetic particles mayhave a broad range of densities, i.e. from substantially less than thatof water, 0.01, to 5 g/mL and preferably from 0.5 to 2 g/mL. Theparticle sizes can range from 0.001 to 100 μm and preferably from 0.01to 10 μm. The concentration of the particles may range broadly from 1 to10,000 μg per mL and preferably is from 5 to 1000 μg per mL.

Desirably the magnetic particles which are used have a low magneticremanence, as described for example EP 0,180,384, so that after themagnetic field is removed from the electrode surface, the particlesdemagnetize and can be swept out of the assay cell. Desirably thedensity, concentration and particle size of the magnetic particles ischosen such that the settling time is at least 0.5 mm/min and desirablyit is above that rate. In operation of the magnetic cell it is oftendesirable to remove the magnet means from the electrode surface prior toinducing electrochemiluminescence in order not to interfere with theoperation of the photomultiplier tube.

Assays

A variety of assays can be performed using the methods of the invention.

An assay was performed as shown in FIG. 3. The PCR products resultingfrom the reaction were labeled with biotin and an ECL label (tris(2,2'bipyridine) Ru II, Ru (bpy)₃ ²⁺). Streptavidin beads captured thebifunctionalized DNA via biotin streptavidin binding and this wasfollowed by washing. The bead bound product was then subjected toanalysis detecting the ECL label.

An assay was performed as shown in FIG. 4. The biotinylated PCR productwas captured on streptavidin beads and the non-biotinylated strandremoved. The bead bound PCR product was then hybridized with an ECLlabeled (Ru (bpy)₃ ²⁺)-oligonucleotide. This was followed by ECLanalysis to detect the label.

An assay was conducted as shown in FIG. 5. The hybrids were captured onstreptavidin beads. This was followed by ECL analysis without washing.

An assay was conducted and the results are shown in FIG. 6. The assaywas for the presence of HPV 16 and 18 using DNA samples isolated fromthe cell lines SiHa and HeLa positive for both virus types andoligonucleotides specific for each virus type. The primers 2PV16, 2PV18were biotinylated and 3PV16, 3PV18 were ECL-labeled-oligonucleotides.The 2/3PV16 and 2/3PV18 oligonucleotides were specific for HPV 16 and 18respectively. The resultant bead captured ECL label was analyzed for ECLusing an analyzer as described in FIG. 1. The results were plotted asECL counts for each sample primer combination.

An assay was conducted and the results are shown in FIG. 7. Theresultant bead bound ECL label was analyzed for ECL using an analyzer asdescribed in FIG. 1. The ECL peak photon counts were plotted againstincreasing concentrations HPV 16 DNA, expressed as a ratio of viralcopies to total cellular DNA copies. The primers used in this analysisfor HPV16 were 1PV16 (biotin label) and 2PV16 (ECL label). DNA used foreach PCR was maintained at a constant 1 μg using calf thymus DNA.

An assay was conducted and the results are shown in FIG. 8. The PCR wasperformed using biotinylated HRP2 with unlabeled HRP1 (for probes 1T24and 1CHR) and biotinylated HRP1 with unlabeled HRP2 (for probes 2T24 and2CHR), generating bead bound single stranded targets for hybridization.The DNA samples were the normal (Placenta) Ha-ras Gene and the mutant(NIH3T3-T24) Ha-ras Gene. The hybridization of the bead bound DNA withECL label-1T24 (1T24), ECL label-2T24 (2T24), ECL label-1CHR (1CHR) andECL label-2CHR (2CHR) was followed by TEMAC washes. Resultant bead boundECL label was analyzed for ECL using an analyzer as described in FIG. 1.Results were plotted as ECL counts for each sample probe combination.

An assay was conducted and the results are shown in FIG. 9. The PCR wasperformed as described in FIG. 8 using only biotinylated HRP2 withunlabeled HRP1 (for probes 1T24 and 1CHR). The probes used were: 1T24and 1CHR containing P³² (1T24-P, 1CHR-P) as controls. With the 1T24 and1CHR containing both P³² and ECL label to determine the effects of theECL label. The samples were washed as earlier with TEMAC. The resultantbead bound P³² was analyzed on addition of scintillation cocktail in ascintillation counter. The results were plotted as P³² counts per secondfor each sample probe combination.

An assay was conducted and the results are shown in FIG. 10. The assaywas performed as described in FIG. 4. The sample was placental DNA andamplification was performed using biotinylated HRP2 with unlabeled HRP1(for probes 1T24 and 1CHR). The resultant PCR product was then sampledto give a set of samples containing differing amounts of product. Thesesets of samples were then hybridized with either probes labeled with P³²(1T24-P32 and 1CHR-P32) or ECL label (1T24-ECL and 1CHR-ECL). Theresults form each studies were then normalized using the average peakvalue from each label for the 90 μl sample. These normalized figuresallow a more effective comparison of signal to background and thecomparative response of the two methods. The inset figure illustratesthe response at the lower level of the dilution curve. The samples werehandled as described earlier. (FIG. 6 and FIG. 8.)

An assay was conducted and the results are shown in FIG. 11. The PCR wasperformed using biotinylated 2PV18 and unlabeled 1PV18 using HeLa DNA(400 copies per cell) using the PCR format illustrated in FIG. 3. Theresultant PCR reaction was then hybridized with the specific probe ECLlabel-3PV18. The hybridization mixture was then added to streptavidincoated beads and the resultant bead bound ECL label was directlyanalyzed for ECL using an ECL analyzer as described in FIG. 1. Theresults were plotted as ECL counts verses HPV18 copies added to the PCR.

EXAMPLES Instrumentation, Materials, and Methods

(1) Instrumentation

A flow-through apparatus, employing three electrodes, as described inFIGS. 1 and 2, was used.

Working Electrode--Au disk, 3 mm diameter

Counter Electrode--Au disk, 3 mm diameter

Reference Electrode--Ag/AgCl

Teflon Gasket (0.15"thick)

Plexiglas Faceplate

Inlet Tubing=0.042" id polypropylene

Aspiration Rates:variable from 0.01 to 5 mL/min

Potentiostat: microprocessor controlled

Luminometer using Hamamatsu R374 PMT (low gain red sensitive tube); PMTVoltage variable 0-1400 V

(2) Materials

(a) ECL Label: Ru(bpy)₃ ²⁺

(b) ECL Buffer: 112 mM KH₂ PO₄, 88 mM K₂ HPO₄ ·3H₂ O, 50 μM NaCl, 6.5 mMNaN₃, 0.8 μM Tirton X-100, 0.4 mM Tween 20, 100 mM tripropylamine in H₂O

(c) ECL Diluent 37.5 mM KH₂ PO₄, 109.2 mM K₂ HPO₄ ·3H₂ O, 151.7 mM NaCl,0.65 mM NaN₃, 0.43 mM bovine serum albumin in H₂ O

(d) Ru(bpy)₃ ²⁺ -NHS: Ru(2,2'-bipyridyl)₂ (4-3-(1,3-dioxolan-2-yl)propyl!-4'-methyl-2,2'-bipyridine)²⁺

(e) Dynal Particles

(i) Dynal M-450 Dynabeads, 4.5 μm diameter superparamagnetic particles,30 mg/mL, obtained from Dynal, 45 North Station Plaza, Great Neck, N.Y.11021

(ii) Dynal M-280 Dynabeads, 2.8 μM diameter superparamagnetic particles,10 mg/mL, obtained for Dynal, 45 North Station Plaza, Great Neck, N.Y.11021

(3) ECL Measurement Cycle (three electrode cell operation)

The ECL measurement cycle consists of three steps:

(1) preconditioning, (2) measuring, and (3) cleaning. Thepreconditioning step involves the application of a voltage triangle waveof 0.0 V to +2.2 V to -1.0 V to +0.6 V at 2.0 V/sec. The measurementstep involves the application of a triangle waveform from +0.6 V to +2.8V to +2.0 V at 1.0 V/s. The cleaning step involves the application of avoltage square wave from +0.0 V to +3.0 V to -0.5 V to 0.0 V. Allvoltages are relative to the Ag/AgCl reference electrode.

Example 1 Apparatus and Method for Collection of Microparticles byGravity

The measurement is conducted in a cell as shown in FIG. 12. Referencesmade to FIG. 12 which depict an apparatus for conducting an assay usingthe force of gravity. The components of the apparatus include atransparent window identified by reference numeral 11, a gasketidentified by reference numeral 12, a block which includes an inlet 13,a working electrode 14, a counterelectrode 15 and an outlet port 16. Theplane of the cell block is horizontal, i.e. perpendicular to thedirection of the earth's gravitational field. Labeled microparticles(Dynal) in an ECL buffer are drawn to the cell by means of a peristalticpump. The pump is turned off after the particles reach the cell. Themicroparticles in the cell chamber fall onto the working electrodesurface. The rate of fall of microparticles is determined to beapproximately constant at 0.5 mm/min over a distance of 10 mm, as shownin FIG. 13. The number of particles to settle is a function of time andrate of fall. The ECL intensity is proportional to the number ofparticles that settle on the working electrode. The number of particlesthat reach the surface, and therefore the ECL intensity is limited bythe height of fluid sample over the working electrode. FIG. 14 shows theECL intensity as a function of deposition time for two cells ofdifferent gasket thicknesses, 0.015 and 0.075 inches, respectively. Bothcells have similar rates of deposition of microparticles but the cellwith a thicker gasket gives an maximum reading which is five timeslarger. The results of an AFP (alpha fetal protein) assay is shown inFIG. 15, comparing the two cells. Again, the cell with the thickergasket produces five times the ECL signal intensity.

Example 2 ECL Apparatus and Method for Deposition of Microparticles.Magnetic Collection using a Sedimentation Cell

A cell for conduct of an assay using magnetic force to cause themicroparticulate to settle is shown in FIG. 16. Reference numeral 21refers to a transparent window, reference numeral 22 to a gasket,reference numeral 23 to the inlet in the cell block, reference numeral24 to the working electrode, reference numeral 25 to the sample outlet,reference numeral 26 to the cell block itself and reference 27 to anelectromagnet.

The plane of the cell block is oriented horizontally. Labeledmicroparticles (Dynal) in ECL buffer are drawn to the cell by means of aperistaltic pump. The pump is turned off after the microparticles reachthe cell. The microparticles in the cell chamber are drawn to theworking electrode by means of a magnetic field generated usingelectromagnet 27 operating at 12 volts and 1.5 amps. By application ofthe electromagnet, the rate of deposition of microparticles is greatlyincreased over that observed when the microparticles settle solely dueto the force of gravity. This is shown in FIG. 17.

Example 3 ECL Apparatus and Method for Deposition of Microparticles.Magnetic Collection using a Collection Cell

An assay is carried out in a cell as described in FIG. 18. Withreference to FIG. 18, reference numeral 31 refers to transparent window,reference numeral 32 to a gasket, reference numeral 33 to an inlet inthe cell block, reference numeral 34 to a working electrode, referencenumeral 35 to the cell block itself, reference numeral 36 to the sampleoutlet and reference numeral 37 to a permanent magnet.

The plane of the cell block is oriented horizontally. Labeledmicroparticles (Dynal) in ECL buffer are drawn to the electrochemicalcell by means of a peristaltic pump. Prior to the sample introduction,permanent magnet 34 is positioned immediately below the workingelectrode/solution interface at a distance of 0.035 inches. As thesample is being drawn to the cell, the microparticles deposit onto anarea over the working electrode, as defined by the area of the magnet.The pump is turned off and the magnetic withdrawn after the entiresample is deposited. The longer the collection time, the more particlesare deposited. Increasing the concentration of particles on the workingelectrode results in an increased ECL intensity as shown in FIG. 19.

Example 4 Use of Magnet for Deposition of Microparticles Magnetic FieldOrientation

Microparticles which are attracted to a magnet whether a permanentmagnet of or electromagnet, align with the orientation of the magneticfield. FIG. 20 depicts magnetic fields and the resultant particlearrangements which are parallel (A) and perpendicular (B) to the surfaceof the working electrode, in the vicinity of that surface.

FIGS. 23 and 24 schematically describe a cell which is equipped with amagnet system which advantageously imposes field lines which are largelyparallel to the plane of the electrode surface. The magnet systemconsists of a plurality of individual permanent or electromagnets whichare stacked and oriented such that the north and south poles of themagnets alternate in the stack. The individual magnets are separated byair or any non-magnetically responsive material. The arrangement asshown in FIGS. 23 and 24 advantageously applies magnetic lines of forceto the region above the working electrode which are nearly horizontal tothe plane of the electrode. This induces an orientation of themagnetically responsive particles in which the particles lie upon thesurface of the electrode and are supplied readily accessible to theelectrochemical energy supplied by the electrode.

The magnet system shown in FIGS. 23 and 24 also is advantageous in thatthe magnetic field lines do not extend a long distance from the magnetstructure. The magnetic field from such a magnet system is not likely,therefore, to induce permanent magnetic behavior on ferromagneticmaterials near the electrode apparatus and will not severely affect theoperation of a photomultiplier tube near the flow cell apparatus.

Example 5 Particle Collection and Concentration by Filtration

Microparticles which are magnetically responsive, non-magneticallyresponsive, and of a wide range of densities can advantageously becollected by filtration upon the surface of a membrane filter. In oneembodiment of the invention, the particles are pumped through a portionof a filter membrane which has pore sizes which are smaller than thediameter of the particles but preferably are substantially smaller thanthe particle diameter and at a sufficiently high surface density suchthat the collection of particles will not cause blockage of the pores.The filter is advantageously largely transparent such that the filter,after collection of the particles, can be placed upon the surface of aworking electrode for the purpose of inducing ECL from the particles andmeasuring the luminescence to measure the quantity of ECL label on theparticles.

In another embodiment, the membrane filter having pore sizes asdescribed above is attached or placed upon the surface of an absorbentmaterial such that capillarity or "wicking" will spontaneously drawfluids containing microparticles through the membrane filter withoutrequiring any apparatus to induce the flow of fluid through the filter.

In the preferred embodiment, the membrane filter, having pore sizes asdescribed above, is coated with a thin film of metal or otherelectronically conductive material such that the surface of the membranecan serve as a working electrode in an ECL apparatus. The conductivefilms are readily applied to the surface of a membrane by methodscommonly used in the fabrication of microelectronic devices, e.g.,thermal evaporation or sputtering. Such a filter-electrode is readilymounted in a flow cell such that the flow-path for the fluid is throughthe filter-electrode. Particles in the stream are trapped by thefilter-electrode and are easily washed in-situ providing for a rapid andsimple means for performing heterogeneous assays without any externalwashing apparatus.

Example 6 Particle Collection and Concentration by Centrifugal Method

The rotary flow cell shown in FIG. 20 provides another means to capturethe complex on the surface of the working electrode in order to measureluminescence. The assay solution 61 is pumped into cell 62 throughrotary seal 63 while a rotational motion is imparted to the cell. Thedenser particles of the complex are concentrated on the surface ofworking electrode 64. While the cell is still rotating the solutionpasses out of the cell. The light output passing through cell window 67is measured by photomultiplier tube 65. The light output is directedfrom the vertical working electrode surface 64 reflecting off curvedmirror surface 66 located at the center of the cell. The cell is thenflushed and cleaned for the next cycle. This may be accomplished withthe cell stopped or rotating.

Example 7 Coating of Particles With Labeled Non-specific Protein atModerate Surface Concentration

30 mg (1 ml) of 4.5 um uncoated magnetically responsive, polystyreneM-450 DYNABEADS (DYNAL, Oslo, Norway) were washed by magnetic seperationwith a 150 mM phosphate buffer pH 7.5 solution using 2 ml/wash. 150 μgof Ru(bpy)₃ ²⁺ -labeled mouse IgG (Jackson Immunochemicals) in 1 ml ofphosphate buffer saline (PBS) with 0.05% thimerasol were added to theparticles. This mixture was allowed to incubate overnight at roomtemperature with rotation. The solution was then magnetically separatedfrom the particles and removed. To block unreacted sites, 1 ml of 3%BSA/PBS with 0.05% sodium azide was added to the particles, and theresultant solution was allowed to incubate 2 hours at room temperature.The particles were washed 5 times (2 ml/wash), and then finallyresuspended in 6 ml of the same buffer for storage.

Example 8 Electrochemiluminescent (ECL) Measurement Using MagneticallyResponsive Particles

Uniform and nonuniform, polymeric and non-polymeric, magneticallyresponsive particles (Dynal, Oslo, Norway; Polysciences, Warrington, Pa.18976; Cortex Biochem, San Leandro, Calif. 94577; Aldrich, Milwaukee,Wis. 53201) were coated with labeled proteins as described in Example 7.The coated particles were washed with ECL buffer three times beforemaking 2 mL of a 300 ug/mL suspension. Using a peristaltic pump, 500 ulof the particle suspension was drawn into the flow cell (Example 3). Asthe particles flowed to the working electrode, they were attracted andconcentrated onto the working electrode surface by a magnet.Electrochemiluminescence using the magnetic particles was measured usinga Hamamatsu R374 photomultiplier tube centered above the flow cell whereparticles had concentrated on the working electrode surface. Table Ishows ECL photoemission levels obtained from the labeled-protein coatedmagnetically responsive particles.

                  TABLE I                                                         ______________________________________                                        ECL Measurements from Different Magnetically Responsive                       Particles                                                                     Particle            Density          ECL                                      Type     Diameter (μm)                                                                         (g/mL)    Material                                                                             Counts                                   ______________________________________                                        Glass    8.0        2.4       soda lime                                                                            2200                                                                   glass                                                    2.0        2.4       soda lime                                                                            8500                                                                   glass                                           Quartz   0.3-3.5    2.5       SiO2   1150                                     Gold     1.0-2.0    19.3      Au     1100                                     ______________________________________                                    

Example 9 Electrochemiluminescent (ECL) Measurement Using NonmagneticParticles

Uniform and nonuniform, polymeric and non-polymeric, non-magneticallresponsive particles (Aldrich, Milwaukee, Wis. 53201; Duke Scientific,Palo Alto, Calif. 94303) were coated with labeled proteins as describedin Example 7. The coated particles were washed with ECL buffer threetimes before making 2 mL of a 300 ug/mL suspension. Using a peristalticpump, 500 ul of the particle suspension was drawn into the flow cell.The coated particles were then concentrated onto the working electrodeby gravitational means as described in Example 1.Electrochemiluminescence using the nonmagnetic particles was measuredwith a Hamamatsu R374 photomultiplier tube centered above the flow cellwhere particles had concentrated on the working electrode surface. TableII shows ECL photoemission levels obtained from the coated nonmagneticparticles.

Example 10 Preparation of Physically Adsorbed Sheep Anti-ThyroidStimulating Hormone (TSH) Coated Dynal Particles (REAGENT I)

1 mL of 4.5 μm uncoated magnetic, polystyrene particles with -OHresidues on their surface (DYNAL, DYNABEADS M-450, DYNAL A.S. Oslo,Norway) was washed by magnetic separation with a 150 mM sodium carbonate/bicarbonate pH 9.6 solution using 2 mL/wash. 0.5 mg of affinitypurified Sheep anti-TSH, HCG scrubbed antibody (CIBA) in 1 mL of thecarb/bicarb solution was added to the particles. This mixture wasincubated overnight at room temperature with mixing. The solution wasthen magnetically separated from the particles and removed. 1 mL of 3%BSA/PBS w/0.05% sodium azide was added and incubated 2 hours at roomtemperature with agitation to block unreacted sites. The particles werewashed 5 times (2 mL/wash) then finally resuspended in 1 mL of the samebuffer for storage. The final concentration of Bead Reagent I was 3% byweight.

                  TABLE II                                                        ______________________________________                                        ECL Measurement from non-magnetically responsive                              Particles by Gravity Collection                                                               Particle                                                      Particle                                                                             Diameter Density               ECL                                     Type   (μm)  (g/mL)   Material     Counts                                  ______________________________________                                        Rhone- 4.0      1.5      Polystyrene Divinyl                                                                        1680                                    Poulenc                  Benzene/ Fe.sub.3 O.sub.4                                   1.5-2.1  1.4      Polystyrene/Fe.sub.3 O.sub.4                                                               462                                     Poly-  1.5-2.1  2.1      Polystyrene/FeO.sub.2                                                                      504                                     sciences                                                                      Dynal  4.5      1.5      Polystyrene/Fe.sub.2 O.sub.3                                                               4200                                    Cortex 1.0-10   1.3      Cellulose/Fe.sub.3 O.sub.4                                                                 125                                            1.0-10   1.8      Polyacrolein/Fe.sub.3 O.sub.4                                                              125                                            1.0-25   1.2      Polyacrylamide/Fe.sub.3 O.sub.4                                                            125                                                              w/ charcoal                                          Nickel 3.0      8.9      Ni           125                                     ______________________________________                                    

Example 11 Preparation of Ouabain-BSA Conjugate (REAGENT II)

ACTIVATION OF OUABAIN

60.4 mg of ouabain octahydrate (Aldrich Cat# 14,193-3) in 6 mL ofdeionized (di) H₂ O (wrapped in foil) was mixed with 87 mg of sodiummetaperiodate (Mallinckrodt Cat# 1139) and the mixture was incubated atroom temperature for 2 hours, rotating. The reaction was terminated bypassing the reaction mixture through Dowex 1×8-50 ion exchange resin(Aldrich Cat# 21,740-9) with diH₂ O. 200 μL 1M sodium phosphate pH 7.2was added to adjust the pH of the solution to 7.0.

CONJUGATION OF ACTIVATED OUABAIN TO BSA

50 mg of activated ouabain (4.6 mL) was then added dropwise to 108 mgbovine serum albumin BSA, Miles Fraction V) in 5 mL 0.15M PBS pH 7.8.This is a 40:1 (OUABIN:BSA) ratio. The reaction was incubated at roomtemperature for 2 hours, mixing, followed by rapid addition of 30 mg ofsodium cyanoborohydride while mixing. Free ouabain and excess sodiumcyanoborohydride were removed by dialysis at 4° C. in 0.15M PBS w/0.05%sodium azide pH 7.8. The Ouabain-BSA Conjugate Reagent II was stored at4° C.

Example 12 Preparation of Physically Adsorbed Ouabain-BSA Coated DynalParticles (REAGENT III)

5 mg of 4.5 μm uncoated magnetic, polystyrene particles with -OHresidues on their surface (DYNAL, DYNABEADS M-450, DYNAL A.S. Oslo,Norway) were washed by magnetic separation with a 150 mM sodiumcarbonate/bicarbonate pH 9.6 solution using 10 mL/wash. 3 mg ofOuabain-BSA conjugate (Conjugate Reagent II) in 5 mL of the carb/bicarbsolution was added to the particles. This mixture was incubatedovernight at room temperature while rotating. The solution was thenmagnetically separated from the particles and removed. 5 mL of 3%BSA/PBS w/0.05% sodium azide was added and incubated 2 hours at roomtemperature, rotating to block unreacted sites. The particles werewashed 5 times (10mL/wash) then finally resuspended in 1 mL of the samebuffer for storage. The final concentration of Bead Reagent III was 3%by weight.

Example 13 Preparation of Ru(bpy)₃ ²⁺ -Labeled Mouse Anti-Digoxin(REAGENT IV)

1 mg of mouse anti-Digoxin (Cambridge Medical Technologies Cat# 200-014Lot A3575) was labeled with Ru(bpy)₃ ²⁺. The monoclonal antibody (MAb)anti Digoxin antibody was buffer exchanged using Centricon 30microconcentrators (Amicon) into 0.15M potassium phosphate buffer, 0.15MNaCl pH 7.8, the final volume being 0.5 mL. Immediately prior to use,0.5 mg of Ru(bpy)₃ ²⁺ -NHS was dissolved with 125 μL of anhydrousdimethyl sulfoxide (Aldrich). To achieve a 25:1 molar ratio of Ru(bpy)₃²⁺ to protein based on molecular weights of 1057 and 150,000respectively, 0.18 mg Ru(bpy)₃ ²⁺ -NHS (45 μL) was added to the proteinsolution while shaking. The reaction tube was incubated in the dark atroom temperature, 30 minutes, while shaking. The reaction was terminatedby the addition of 25 μL of 1M glycine and incubated for 10 minutes. Thereaction mixture was purified by passage through a Sephadex G-25 column(1×20 cm in 0.15M potassium phosphate, 0.15M NaCl with 0.05% sodiumazide pH 7.2). The Ru(bpy)₃ ²⁺ -labeled mouse anti-digoxin fractionswere collected and pooled. The labeled protein was determined to have 12labels per protein molecule.

Example 14 Preparation of Ru(bpy)₃ ²⁺ -Labeled Mouse Anti-ThyroidStimulating Hormone (TSH) (REAGENT V)

0.5 mg of mouse anti-TSH (CIBA) was labeled with Ru(bpy)₃ ²⁺. The MAbanti TSH antibody was buffer exchanged using Centricon 30microconcentrators (Amicon) into 0.15M potassium phosphate buffer, 0.15MNaCl pH 7.8, the final volume being 0.35 mL. Immediately prior to use,0.5 mg of Ru(bpy)₃ ²⁺ -NHS was dissolved in 75 μL of anhydrous dimethylsulfoxide (Aldrich). To achieve a 50:1 molar ratio of Ru(bpy)₃ ²⁺ labelto protein based on molecular weights of 1057 and 150,000 respectively,0.176 mg Ru(bpy)₃ ²⁺ -NHS (26.4 μL) was added to the protein solutionwhile shaking. The reaction tube was incubated in the dark at roomtemperature, 30 minutes, while shaking. The reaction was terminated bythe addition of 25 μL of 1M glycine and incubated for 10 minutes. Thereaction mixture was purified by passage through a Sephadex G - 25column (1×20 cm in 0.15M potassium phosphate, 0.15M NaCl with 0.05%sodium azide pH 7.2). The Ru(bpy)₃ ²⁺ -labeled mouse anti-TSH fractionswere collected and pooled. The labeled protein was determined to have 14labels per protein. Reagent V.

Example 15 One Step Separation Sandwich Assay for Thyroid StimulatingHormone (TSH)

100 μL serum calibrators (London Diagnostics TSH LumiTAG Kit), 25 μLRu(bpy)₃ ²⁺ -labeled mouse anti-TSH (Reagent V) in ECL buffer and 25 μLSheep anti-TSH-DYNAL particles (Reagent I) in ECL buffer were combinedand incubated in polypropylene tubes for 15 minutes, at roomtemperature, with mixing. The particles were then washed by magneticseparation and then resuspending the particles in 500 μL of ECL buffer.This wash procedure was repeated two additional times. Finally, theparticles were resuspended in 1 mL of ECL buffer. Theelectrochemiluminescence (ECL) for each sample was read as described inExample 3. The ECL counts are directly proportional to the concentrationof analyte present in the sample (increasing counts as the concentrationof analyte increases). Table III demonstrates a representative assaycurve.

                  TABLE III                                                       ______________________________________                                        One-Step Separation Sandwich Assay: Detection of TSH                          TSH                                                                           Concentration   ECL Counts                                                    (μIU/mL)     (Duplicate Samples)                                           ______________________________________                                        0.00            1918     1885                                                 0.05            2584     2530                                                 0.10            3365     3288                                                 0.50            8733     8652                                                 2.50            35688    35347                                                10.0            125316   136994                                               25.0            300248   288272                                               50.0            549034   564948                                               ______________________________________                                    

Example 16 One Step Non Separation Sandwich Assay for ThyroidStimulating Hormone (TSH)

100 μL serum calibrators (London Diagnostics TSH LumiTAG Kit), 25 μLRu(bpy)₃ ²⁺ -labeled mouse anti-TSH (Reagent V) in ECL buffer and 25 μLSheep anti-TSH-DYNAL particles (Reagent I) in ECL buffer were combinedand incubated in polypropylene tubes for 15 minutes, at roomtemperature, with mixing. Prior to reading results, 1 mL of ECL bufferwas added. The electrochemiluminescence (ECL) for each sample was readas described in Example 3. The ECL counts are directly proportional tothe concentration of analyte present in the sample (increasing counts asthe concentration of analyte increases). Table IV demonstrates arepresentative assay curve.

                  TABLE IV                                                        ______________________________________                                        One-Step Non-Separation Sandwich Assay:                                       Detection of TSH                                                              TSH                                                                           Concentration    ECL Counts                                                   (μIU/mL)      (Duplicate Samples)                                          ______________________________________                                        0.00             2610    2769                                                 0.05             2870    2894                                                 0.10             2970    2950                                                 0.50             3473    3403                                                 2.50             5588    5495                                                 10.0             13051   13139                                                25.0             26468   27306                                                50.0             47104   48664                                                ______________________________________                                    

Example 17 Two Step Separation Competitive Assay for Digoxin

50 μL serum calibrator (TDx Assay, Abbott Labs, Chicago, Ill.) and 25 μLRu(bpy)₃ ²⁺ -labeled mouse anti-Digoxin (Reagent VI) in ECL buffer, werecombined and incubated 20 minutes at room temperature with mixing. 25 μLOuabain-BSA-DYNAL particles (Reagent III) in ECL buffer was added andincubated an additional 20 minutes, at room temperature, with mixing.The particles were then washed by magnetic separation and thenresuspending the particles in 500 μL of ECL buffer. This wash procedurewas repeated two additional times. Finally, the particles wereresuspended in 1 mL of ECL buffer. The electrochemiluminescence (ECL)for each sample was read as described in Example 3. The ECL counts areinversely proportional to the concentration of analyte present in thesample (decreasing counts as the concentration of analyte increases).Table V demonstrates a representative assay curve.

                  TABLE V                                                         ______________________________________                                        Two-Step Separation Competitive Assay:                                        Detection of Digoxin                                                          Digoxin                                                                       Concentration    ECL Counts                                                   (ng/mL)          (Duplicate Samples)                                          ______________________________________                                        0.0              22031   21154                                                0.5              13367   13638                                                1.0              9506    9607                                                 2.0              5244    5129                                                 3.0              2959    2994                                                 5.0              1581    1631                                                 ______________________________________                                    

Example 18 Two Step Non Separation Competitive Assay for Digoxin

50 μL serum calibrator (TDx Assay, Abbott Labs, Chicago, Ill.) and 25 μLRu(bpy)₃ ²⁺ -labeled mouse anti-Digoxin (Reagent VI) in ECL buffer, werecombined and incubated 20 minutes at room temperature with mixing. 25 μLOuabain-BSA-DYNAL particles (Reagent III) in ECL buffer was added andincubated an additional 20 minutes, at room temperature, with mixing.Prior to reading, the particles were resuspended in 1 mL of ECL buffer.The electrochemiluminescence (ECL) for each sample was read as describedin Example 3. The ECL counts are inversely proportional to theconcentration of analyte present in the sample (decreasing counts as theconcentration of analyte increases). Table VI demonstrates arepresentative assay curve.

                  TABLE VI                                                        ______________________________________                                        Two-Step Non-Separation Competitive Assay:                                    Detection of Digoxin                                                          Digoxin                                                                       Concentration    ECL Counts                                                   (ng/mL)          (Duplicate Samples)                                          ______________________________________                                        0.0              42051   39643                                                0.5              28721   28074                                                1.0              22190   21364                                                2.0              14660   14542                                                3.0              11315   11893                                                5.0               9161    8945                                                ______________________________________                                    

Example 19 Two Step Non Separation Competitive Assay for Digoxin Using aRead Cycle With Additional Washing of Final Reaction Sample on theElectrode

50 μL serum calibrator (TDx Assay, Abbott Labs, Chicago, Ill.) and 25 μLRu(bpy)₃ ²⁺ -labeled mouse anti-Digoxin (Reagent VI) in ECL buffer, werecombined and incubated 20 minutes at room temperature with mixing. 25 μLOuabain-BSA-DYNAL particles (Reagent III) in ECL buffer was added andincubated an additional 20 minutes, at room temperature, with mixing.Prior to reading, the particles were resuspended in 1 mL of ECL buffer.The electrochemiluminescence (ECL) for each sample was read as describedin Example 3. The ECL counts are inversely proportional to theconcentration of analyte present in the sample (decreasing counts as theconcentration of analyte increases). Table VII demonstrates arepresentative assay curve.

                  TABLE VII                                                       ______________________________________                                        Two-Step Separation Competitive Assay:                                        Detection of Digoxin                                                          Digoxin                                                                       Concentration    ECL Counts                                                   (ng/mL)          (Duplicate Samples)                                          ______________________________________                                        0.0              42613           35309                                        0.5              24211           24168                                        1.0              17561           17206                                        2.0              10491            9909                                        3.0               6712            7145                                        5.0               4680            4603                                        ______________________________________                                    

Example 20 Oligonucleotide Synthesis

The oligonucleotides were made on an Applied Biosystems automatedoligonucleotide synthesizer using the β-cyanoethyl phosphoramidite (1).Oligonucleotide amino modifications to the 5' end occurred at the lastcoupling step, and at the 3' end by using a modified solid phase(controlled pore glass). Clontech (San Diego, Calif.) supplied the aminomodifiers. The resulting 5' modified oligonucleotides all contain a sixcarbon spacer arm to the amino group designated (C6, NH2). The 3'modified oligonucleotides all contain a three carbon spacer to the aminogroup. Oligonucleotides constructed, their modifications and utility aredescribed below.

Oligonucleotides for the HPV study were directed to the E6 region aspreviously described (2).

The oligonucleotide sequences were as follows:

HPV 16; 1PV16 5' (C6, NH2) TTAGTGAGTATAGACATTATTGTTATAGTT;

2PV16 5' (C6, NH2) CAGTTAATACACCTAATTAACAAATCACAC;

3PV16 5' (C6, NH2) ACAACATTAGAACAGCAATACAACAAACCG;

HPV18; 1PV18 5' (C6, NH2) TTAGAGAATTAAGACATTATTCAGACT;

2PV18 5' (C6, NH2) CACCGCAGGCACCTTATTAATAAATTGTAT;

3PV18 5' (C6, NH2) GACACATTGGAAAAACTAACTAACACTGGG.

These oligonucleotides enable the PCR generation of various fragments;3PB16 or 3PV18 with 2PV16 or 2PV18 respectively form a 62 bp fragment;1PV16 with 2PV16 form a 100 bp fragment; 1PV18 with 2PV18 form a 103 bpfragment. It will be appreciated that the 3PB16 and 3PV18oligonucleotides can also be used as probes hybridizing to the productsfrom the PCR reaction of 1PV16 with 2PV16 and 1PV18 with 2PV18,hybridizing to the strand produced by the oligonucleotides 2PV16 and2PV18 within the PCR.

Oligonucleotides for the Ha-ras point mutation assays were as follows:

HRP1 5' (C6, NH2) GCGATGACGGAATATAAGCTGGTGGTGGTG;

HRP2 5' (C6, NH2) TTCTGGATCAGCTGGATGGTCAGCGCACTC;

These two oligonucleotide primers direct the PCR synthesis of an 80 bpfragment. The sequences of the probes used for this point mutation studywere as follows:

1T24 5' (C6, NH2) GGCGCCGTCGGTGTGGGCAA;

1CHR 5' (C6, NH2) GGCGCCGGCGGTGTGGGCAA;

2T24 5' (C6, NH2) TTGCCCACACCGACGGCGCC;

2CHR 5' (C6, NH2) TTGCCCACACCGCCGGCGCC.

Aside from these sequences we also synthesized the above 1CHR and 2T24sequences without the 5' amino modification but with a 3' amino group.These 3' amino modified oligonucleotides were labeled with the ECL labeland used in hybridizations. The site of the mutation/mismatch isindicated by the nucleotide in bold. The probes 1T24 and 1CHR hybridizeto the strand produced by oligonucleotide HRP2 within the PCR. Theprobes 2T24 and 2CHR hybridize to the strand produced by oligonucleotideHRP1 within the PCR. Oligonucleotides JK8 and JK8C for coupling toparticles:

JK8 5' (C6, NH2)GTCCAATCCATCTTGGCTTGTCGAAGTCTGA

JK8C 5' (C6, NH2)TCAGACTTCGACAACCCAAGATGGATTGGA1C.

These two sequences are derived from aequorin sequences and arecomplementary to each other.

JK7 5'TCAGACTTCGACAA(NH2)CCCAAGATGGATTGGA:

This oligonucleotide was amino modified using an amino modifier fromClontech (San Diego, Calif.) which allows amino modifications within thesequence. JK7 was labeled using the Ru(bpy)₃ ²⁺ -label.

Oligonucleotide probe for aequorin RNA generated by in vitrotranscription:

T35 5' (NH2)GATTTTTCCATTGTGGTTGACATCAAGGAA;

this oligo was labeled with both biotin and Ru(bpy)₃ ²⁺ -label.

For the detection of Escherichia coli DNA we synthesizedoligonucleotides specific for the Trp E/D region of the genome (3) asfollows:

TRP.CO3 5' (C6,NH2)GCCACGCAAGCGGGTGAGGAGTTCC(NH2);

this sequence was labeled with Ru(bpy)₃ ²⁺ -label and

TRP.CO4 5' (C6,NH2)GTCCGAGGCAAATGCCAATAATGG

was labeled with biotin as described below.

Example 21 Labeling Oligonucleotides

All the synthetic oligonucleotides were purified to remove anycontaminating amino groups by gel filtration on a Biogel P6 (BioRadLabs) column. Biotin was introduced via the 5'-amino group of the PCRprimers using NHS-biotin (Clontech, San Diego, Calif.) (4). Ru(bpy)₃ ²⁺-NHS was introduced via the amino group of the modified oligonucleotidesas follows. The oligonucleotides (0.1 μmole) in 100 μl of PBS (pH 7.4)were reacted with 5 μmole of Ru(bpy)₃ ²⁺ -label dissolved in DMSOovernight at room temperature in the dark. Oligonucleotides wererecovered from these labeling reactions by ethanol precipitation. Recentstudies have demonstrated the ability to effectively label (>80%) using0.5 μmole of the Ru(bpy)₃ ²⁺ -label (data not shown).

The labeled oligonucleotides were further purified by HPLC on a reversephase Vydac C-18 semiprep column with mobile phases of A) 100 mMtetraethylammonium acetate pH 7.0 and B) 50% A) and 50% acetonitrile,running the gradient from 20% to 40% of B.

Probes 1CHR and 1T24 were also labeled with ³² p using T4 polynucleotidekinase using established methods generating probes with a specificactivity of 77,000 cpm/ng (5).

Example 22 Preparation of Nucleic Acid Magnetic Particles

Dynal M 450 particles were activated with 2-fluoro-1-methylpyridiniumtoluene-4-sulfonate using standard procedures (6). These activatedparticles were then reacted with oligonucleotides JK8 and JK8C. To 100mg of activated Dynal particles were added 33 nmoles of oligonucleotidein 650 μl of 0.1M NaHCO₃ followed by incubation for 3 hours with mixing.The particles were blocked by the addition of ethanolamine (4 mL, 0.1M).The coupled particles were mixed with 0.5 mg/mL single stranded salmonsperm DNA in ECL buffer, washed 4-5 times into ECL buffer andresuspended at 10 mg/mL in ECL buffer containing 100 μg/mL singlestranded salmon sperm DNA.

Example 23 Preparation of Streptavidin Magnetic Particles I

Dynal M 450 particles were activated with 2-fluoro-1-methylpyridiniumtoluene-4-sulfonate using standard procedures (6). The activatedparticles were then reacted with streptavidin (Sigma Ltd). Activatedparticles (50 mg) were washed with 0.1M NaHCO₃ followed by the additionof streptavidin (1.5 mg) and reacted overnight. The particles wereblocked by the addition of ethanolamine (4 mL, 0.1M). The coupledparticles were mixed with 0.5 mg/mL single stranded salmon sperm DNA inECL buffer, washed 4-5 times into ECL buffer and resuspended at 10 mg/mLin ECL buffer containing 100 μg/mL single stranded salmon sperm DNA. Thestreptavidin particles from Dynal M-280 also proved valuable but gavelower signals with the current assay sequence. For immunoassayapplications particles were blocked with BSA after antigen or antibodycoupling using the buffers used for passive coating.

Example 24 Preparation of Streptavidin Magnetic Particles II

To 15 mg of BSA (in 2-3 mL PBS), 105 ul of dimethylsulfoxide containing50 mg/mL of biotin-x-NHS (Clontech, San Diego, Calif. 5002-1) was addedfollowed by mixing and incubation at room temperature for 30 minutes.The reaction was stopped by adding 30 ul of 1M glycine and incubation atroom temperature for 10 minutes. The reaction mix was purified by gelfiltration chromatography (Biorad, Bio-Gel P6, ). This biotin-BSA wasfiltered using 0.2 um syringe. 5 mg biotin-BSA in 10 mL of 0.2M sodiumcarbonate/bicarbonate buffer pH 9.6 (carbonate/bicarbonate) buffer wasadded to 300 mg of Dynabeads washed with carbonate/bicarbonate (Dynal14002). This mixture was Vortexed, and incubated overnight at roomtemperature with mixing. These particles were magnetically separatedfollowed by the addition of 10 mL ECL diluent and 100 ul tRNA (10mg/mL). This mixture was incubated for 3-4 hours at room temperaturewith mixing. These particles were washed once with 10 mL of ECL diluentand resuspended in 10 mL of ECL diluent and 100 ul tRNA (10 mg/mL). Thismixture was mixed and incubated at 2°-6° C. overnight to stabilizeproteins on particles. The particles were magnetically separated andsuspended in 10 mL of PBS containing 15 mg of streptavidin (ScrippsS1214) followed by mixing for one hour. The particles were washed 4times in 10 mL ECL diluent, with 5 minutes mixing for each wash. Theparticles were finally resuspended in 29.7 mL of ECL diluent and 300 ultRNA (10 mg/mL) to a final concentration of 10 mg/ mL particles+100 ug/mL tRNA.

Example 25 Detection of Immobilized DNA on Particles by HybridizationWith ECL DNA Probes

The ability to detect ECL after hybridization to particles wasdemonstrated by the hybridization of particles coupled to JK8 and JK8Cwith Ru(bpy)₃ ²⁺ -label oligonucleotide JK7. Individual lots ofparticles (300 μg) in ECL buffer were mixed with 50 μl of ECL buffercontaining 12.5, 6.3, 3.01, and 1.5 fmoles of labeled JK7. Thesemixtures were hybridized for 4hours at 52° C. followed by washing with 1mL of ECL buffer and resuspension in 830 μl of ECL buffer. These sampleswere analyzed as described in Example 1. The probe JK7 is complementaryto the JK8 sequence and not complementary to JK8C sequence.

                  TABLE VIII                                                      ______________________________________                                        Particles   Probe amount (fmoles)                                                                       ECL counts                                          ______________________________________                                        JK8         12.5          5085                                                            6.3           3035                                                            3.01          1345                                                            1.5           657                                                 JK8C        12.5          451                                                             6.3           345                                                             3.01          256                                                             1.5           212                                                 ______________________________________                                    

The results shown in Table VIII demonstrate the ability to detect byspecific hybridization the presence of specific sequences directlyimmobilized on the surface of particles by ECL.

Example 26 RNA Assay Based on Bead Bound ECL

Dynal M450 particles were coated with antibody specific for RNA/DNAantibodies (7) following standard procedures (Example 10). Specific RNAspecies were generated using plasmids derived from our cloned aequoringene (8). In brief, the plasmid pA5' was cut with EcoRI purified andsubjected to in vitro transcription using T3 RNA polymerase generatingT3-RI RNA (negative RNA). Also plasmid pA5' was cut with BamHI purifiedand subjected to in vitro transcription using T7 RNA polymerasegenerating T7-Bam RNA (positive RNA). These two RNA species thusrepresent two complementary RNA species. These RNA species were purifiedby extraction with an equal volume of phenol:chloroform (50:50) followedby chloroform extraction and precipitation of the supernatant using 2.5vols of ethanol. The amount of RNA was determined using gelelectrophoresis and spectrophotometry. These methods are wellestablished and known to those skilled in the art (9).

Streptavidin was labeled with Ru(bpy)₃ ²⁺ -label using establishedmethods using a 25:1 molar excess of Ru(bpy)₃ ²⁺ -label overstreptavidin (Example 13). This labeled streptavidin was purified usingan iminobiotin column following established methods (10). Thestreptavidin was estimated to contain 10 Ru(bpy)₃ ²⁺ -labels perstreptavidin tetramer. This labeled streptavidin was then complexed withbiotinylated T35, this was achieved using a one to one mix ofoligonucleotide to labeled streptavidin. Specifically 20 pmoles of eachwere mixed in a final volume of 15 μl of ECL buffer and incubated overnight at 4° C. to form the labeled streptavidin-oligonucleotide (SA-T35)complex. The samples of positive and negative RNA (10 ng) werehybridized to 2 μl of the SA-T35 complex (one step assay) or 25 ng ofthe biotinylated T35 (two step assay). Samples were made up to 50 μl andhybridized for 3 hours at 50° C. followed by the addition of 200 μg ofanti DNA/RNA antibody coated particles in 20 μl of PBS 0.1% BSA. Thismixture was mixed at room temperature for 1 hour followed by two washesin ECL buffer. Samples from the hybridization with the SA-T35 complexwere resuspended in 530 μl of ECL buffer and analyzed as described inExample 1. Those samples from the hybridization with biotinylated T35alone were then incubated with 50 pmoles of labeled streptavidin andincubated for 1 hr with mixing followed by two washes in ECL buffer.Samples from the hybridization were resuspended in 530 μl of ECL bufferand analyzed as described in Example 1. The results are presented inTable IX.

                  TABLE IX                                                        ______________________________________                                        ASSAY       RNA      ECL COUNTS (average)                                     ______________________________________                                        One Step    Positive 815                                                                  Negative  91                                                      Two Step    Positive 1123                                                                 Negative 194                                                      ______________________________________                                    

Example 27 Polymerase Chain Reactions

Polymerase chain reaction's were performed essentially as described (11,12, 13). Reactions were typically of 100 μl unless otherwise stated. PCRcarried out in the asymmetric mode directed incorporation of theRu(bpy)₃ ²⁺ -label, using 5 pmoles of the biotinylated oligonucleotideand 50 pmoles of Ru(bpy)₃ ²⁺ -label oligonucleotide. We ran the assayfor the Ha-ras point mutation under identical conditions but without theRu(bpy)₃ ²⁺ -labeled oligonucleotide. Also, we ran the non-separationHPV assay asymmetrically but making use of a ten fold excess of thebiotinylated oligonucleotide typically 40 pmoles. The thermocyclerconditions were as follows, for the direct incorporation HPV 18 and 16assay, the profile was 93° C. 1 sec, 50° C. 1 sec, 60° C. 2 min; for theHa-ras point mutation assay 93° C. 1 sec, 69° C. 2 min; for thenon-separation HPV assay 93° C. 10 sec, 50° C. 30 sec, 60° C. 2 min. Thecycle numbers for these PCR runs were from 30 to 40 depending on theassay and the required degree of sensitivity.

Example 28 DNA-PROBE ASSAY FORMAT I. Detection and Quantitation ofHumanPapilloma Virus PCR Products by Enzymatic Incorporation.

Following PCR using direct incorporation of the Ru(bpy)₃ ²⁺ -labeloligonucleotide, the whole reaction mixture (90-100 μl) was added to 600μg of streptavidin coupled magnetic particles I, followed by incubationfor 20 min at room temperature with shaking. The solid phase in thesesamples was separated using magnetic racks, washed twice with ECLbuffer, resuspended in 530 μl of ECL buffer and then analyzed forelectrochemiluminescence as described in Example 1. FIG. 3 illustratesthis assay format. The results for this assay format were demonstratedwith human papilloma virus samples (2,14). Specificity studies of thedirect incorporation of Ru(bpy)₃ ²⁺ -label-oligonucleotides intobiotinylated PCR products made use of the closely related virus typesHPV16 and HPV18. Assay for the presence of HPV 16 and 18 using DNAsamples positive for both virus types and oligonucleotides specific foreach virus type. The primers were as follows 2PV16, 2PV18 werebiotinylated and 3PV16, 3PV18 were Ru(bpy)₃ ²⁺ -label-oligonucleotides.The 2/3PB16 and 2/3PV18 oligonucleotides were specific for HPV 16 and 18respectively. The resultant bead-captured Ru(bpy)₃ ²⁺ -label wasanalyzed for ECL as described in Example 1. The results were plotted asECL counts for each sample primer combination see FIG. 6.

To demonstrated the quantitative nature of our assay format a standardcurve of directly incorporated Ru(bpy)₃ ²⁺ -label and biotinylatedoligonucleotides into HPV16 PCR products was generated. The resultantbead-bound Ru(bpy)₃ ²⁺ -label was analyzed for ECL as described inExample 1. The ECL peak photon counts were plotted verses increasingconcentrations HPV 16 DNA, expressed as a ratio viral copies to totalcellular DNA copies. The primers used in this analysis HPV16 were 1PV16(biotin label) and 2PV16 Ru(bpy)₃ ²⁺ -label). DNA used for each PCR wasmaintained at a constant 1 μg using calf thymus DNA. The results forthis standard curve are shown in FIG. 5. These results of specificityand quantitation for this format demonstrates the ability of these ECLlabels to produce simple and rapid DNA based assays. It alsodemonstrates the ability of the label to interface readily in enzymereactions without interfering in the enzymatic process.

Example 29 DNA-PROBE ASSAY FORMAT II. Detection and Determination ofPoint Mutations in the Human Ha-ras Oncogene PCR Amplified Product

We carried out the PCR reactions of Ha-ras genes using oligos HRP1 andHRP2. Using biotinylated HRP1 with unlabeled HRP2 the resulting PCRproduct can hybridize to Ru(bpy)₃ ²⁺ -label probes, 2CHR and 2T24.Conversely using biotinylated HRP2 with unlabeled HRP1 the resulting PCRproduct can hybridize to Ru(bpy)₃ ²⁺ -label probes, 1CHR and 1T24 . TheDNA used was human placental (normal) and mouse NIH3T3 cell DNA,transfected with the mutant Ha-ras gene from the bladder carcinoma T24(15).

The assay protocol was as follows; 90 μl of PCR reaction mixture wasadded to 600 μg of streptavidin coupled magnetic particles I, followedby incubation at room temperature for 30 min. The solid phase in thesesamples was separated using magnetic racks, washed with 50 mM NaOH,washed with hybridization buffer (0.9M NaCl, 50 mM NaPO4, pH 7.7, 5 mMEDTA, 0.1% w/v ficoll, 0.1% w/v polyvinylpyrrolidone, 0.1% w/v bovineserum albumin) and resuspended in hybridization buffer containing 10μg/mL of the Ru(bpy)₃ ²⁺ -label-oligonucleotide. These samples werehybridized for 15 min at 66° C.

The solid phase was separated using magnetic racks, washed twice with0.9M NaCl, 50 mM NaPO4, pH 7.7, 5 mM EDTA, washed with 3Mtetramethylammonium chloride, 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.025%triton X-100 at room temperature once and at 66° C. twice for 20 mineach. The solid phase was washed with ECL buffer three times,resuspended in 530 μ1 ECL buffer and electrochemiluminescence detectedas described in Example 1. FIG. 4 illustrates this assay format.

The assays for Ha-ras PCR products using P³² labeled probes were similarto those using Ru(bpy)₃ ²⁺ -label except the solid phase was finallyresuspended in 250 μl of ECL buffer. These suspended samples were thentransferred to 5 mL of scintillation fluid and counted on a BeckmanLS-100C liquid scintillation counter.

In FIG. 8 we show data from a point mutation assay for the Ha-rasoncogene. The PCR was performed as illustrated in FIG. 4 usingbiotinylated HRP2 with unlabeled HRP1 (for probes 1T24 and 1CHR) andbiotinylated HRP1 with unlabeled HRP2 (for probes 2T24 and 2CHR),generating bead-bound single stranded targets for hybridization. The DNAsamples were the normal (Placenta) Ha-ras Gene and the mutant(NIH3T3-T24) Ha-ras Gene. The hybridization of the bead-bound DNA withRu(bpy)₃ ²⁺ -label-1T24 (1T24 ), Ru(bpy)₃ ²⁺ -label-2T24 (2T24),Ru(bpy)₃ ²⁺ -label-1CHR (1CHR) and Ru(bpy)₃ ²⁺ -label-2CHR (2CHR) wasfollowed by TEMAC washes. Resultant bead-bound Ru(bpy)₃ ²⁺ -label wasanalyzed for ECL as described in Example 1. Results were plotted as ECLcounts for each sample probe combination. The results (FIG. 8) were asexpected with the normal probes hybridizing well to the normal DNA (seethe CHR probes) and the mutant probes hybridizing to the mutant gene(see the T24 probes). It was of interest that these probes did not allperform equivalently. To investigate this apparent anomaly we studiedthese probes further using P³² labeled probes with and without Ru(bpy)₃²⁺ -label. This Evaluation of the specificity of the Ru(bpy)₃ ²⁺ -labelprobes using P³² labeled probes for the Ha-ras oncogene was carried outas follows. The PCR was performed as described in FIG. 8 using onlybiotinylated HRP2 with unlabeled HRP1 (for probes 1T24 and 1CHR). Theprobes used were: 1T24 and 1CHR containing P³² (1T24-P, 1CHR-P) ascontrols. With the 1T24 and 1CHR containing both P³² and Ru(bpy)₃ ²⁺-label to determine the effects of the Ru(bpy)₃ ²⁺ -label. The sampleswere washed as earlier with TEMAC. The resultant bead-bound P³² wasanalyzed on addition of scintillation cocktail in a scintillationcounter. The results were plotted as P³² counts per second for eachsample probe combination (see FIG. 9). This result demonstrated that theP³² probes and the Ru(bpy)₃ ²⁺ -labeled probes function equivalently andthat problems with the probe specificity are due to the specific probesequences used. To further demonstrate this equivalence of our Ru(bpy)₃²⁺ -label and P³² we conducted an comparison between these labeledprobes. The amplification was performed as previously described usingplacental DNA, using biotinylated HRP2 with unlabeled HRP1 (for probes1T24 and 1CHR). The resultant PCR product was then sampled to give a setof samples containing differing amounts of product. These sets ofsamples were then hybridized with either probes labeled with P³²(1T24-P32 and 1CHR-P32) or Ru(bpy)₃ ²⁺ -label (1T24-Ru(bpy)₃ ²⁺ and1CHR-Ru(bpy) ₃ ²⁺). The results from each study were then normalizedusing the average value from each label for the 90 μl of sample. Thesenormalized figures allow a more effective comparison of signal tobackground and the comparative response of the two methods. The inset toFIG. 10 illustrates the response at the lower level of the dilutioncurve. The samples were handled as described earlier (FIG. 8 and FIG.9). Results in FIG. 10 demonstrated the equivalency of the two labelswith indications of a better response from our Ru(bpy)₃ ²⁺ -labeledprobe. These studies demonstrated the ability of Ru(bpy)₃ ²⁺ -labeledprobes to function as well as P³² labeled probes in their ability todiscriminate single base changes in sample DNA. This evidence indicatesthat the Ru(bpy)₃ ²⁺ -label does little to affect the properties of thelabeled probe in hybridization reactions.

Example 30 DNA-PROBE ASSAY FORMAT III. Detection and Quantitation ofHuman Papilloma Virus PCR Products in a Non-Separation Assay

For the non-separation assay on HPV 18, we performed an asymmetric PCRreaction with an excess of the biotinylated primer. This PCR reactiongenerates an excess of biotinylated single-stranded DNAs now availablefor direct hybridization by the Ru(bpy)₃ ² +label-probes.

For hybridization, we added 1000 ECL counts of Ru(bpy)₃ ²⁺-label-oligonucleotide (.sup.˜ 2 ng) specific for the HPV gene amplifiedto 15 μl of the PCR after completion of the amplification followed byincubation for 15 min at 50° C. To this hybridization mixture we added60 μl of ECL buffer containing 600 μg of streptavidin coupled magneticparticles I and incubated with shaking at room temperature for 15 min.The sample volume was increased to 530 μl by addition of ECL bufferfollowed by detection of electrochemiluminescence as described inExample 1. FIG. 5 illustrates this assay format. To demonstrate thisnon-separation assay we ran a standard curve of HPV18 DNA. The PCR wasperformed using biotinylated 2PV18 and unlabeled 1PV18 using HeLa DNA(14). The resultant PCR reaction was then hybridized with the specificprobe Ru(bpy)₃ ²⁺ -label-3PV18. The hybridization mixture was then addedto streptavidin coated particles and the resultant bead-bound Ru(bpy)₃²⁺ -label was directly analyzed for ECL as described in Example 1. Theresults were plotted as ECL counts verses HPV18 copies added to the PCRwith a control of the ras oligonucleotide probe (see FIG. 11). Theseresults demonstrate the ability to generate rapid non-separation assaysfor nucleic acid sequences based on the properties of the ECL assaysystem.

Example 31 Assay for Specific Genomic DNA Sequences

The assay format described here makes use of two oligonucleotides, bothhybridize to the same DNA strand next to each other, one probe allowscapture; the other labels the complex (sandwich hybridization). Thisassay was demonstrated using E. coli DNA and probes specific for the trpE/D gene region. The E. coli DNA was prepared following standardprotocols (16). The salmon sperm control DNA was purchased from SigmaLtd. To the samples of DNA were added 14 μl of hybridization buffer(10×PBS, 10 mMEDTA and 0.7% SDS), 2 ng of biotin labeled TRP.CO4 and 5ng of Ru(bpy)₃ ²⁺ -label TRP.CO3. These samples were made up to 100 μlwith water. The samples were heated to 97° C. and incubated at 97° C.for 10 min, cooled to 50° C. and hybridized for 2 hrs. To these sampleswe added 20 μl of streptavidin coated magnetic particles II and mixedfor 2 hrs at room temperature. The particles were then washed 4 times inECL buffer resuspended in 500 μl of ECL buffer and analyzed as describedin Example 3. The positive DNA is E. coli and the negative DNA is salmonsperm. The results are shown in Table X.

                  TABLE X                                                         ______________________________________                                        DNA          Amount  Average ECL counts                                       ______________________________________                                        Positive     10      184                                                                   25      257                                                                   50        266.5                                                  Negative     10       87                                                                   25       70                                                                   50       75                                                      ______________________________________                                    

These results demonstrated the ability of the ECL assay system tofunction in the detection of a genomic gene in E. coli using a sandwichhybridization assay format on non amplified DNA.

rom our RU(bpy)₃ ²⁺ -labeled probe. These stud

Example 32 Particle Concentration on Evanescent-Wave FluorescenceDetectors

Concentration of labeled complex on a detection surface can be used toincrease sensitivity of assays using evanescent wave detectors. Suchdetectors may use either optical fibers or planar optical waveguides tocarry light from a light source to the fluid environment. The light isreflected through the waveguide or optical fiber by total internalreflection (TIR) which occurs when an incident light beam strikes aninterface between a dielectric medium of high refractive index (n₁) andone of lower refractive index (n₂). When the incident angle of the lightbeam is greater than the critical angle, θ₀ =sin⁻¹ (n₂ /n₁), then thelight is 100% internally reflected at the interface. In opticalwaveguides and optical fibers, light travels with an incidence anglegreater than this critical angle, and propagates through the medium bytotal internal reflectance. FIG. 22 depicts the TIR propagation in awaveguide or optical fiber.

Although the light ray is totally reflected at each interaction with theinterface, the electromagnetic field is not zero outside the medium.Physical requirements of continuity across an interface require that theelectromagnetic field decay exponentially as it penetrates outside thefiber or waveguide into the external environment. This field is calledthe evanescent field and is capable of exciting fluorophores tofluoresce. The decay rate of the evanescent field depends on theincident wavelength, refractive indices n₁ and n₂, and the angle ofincidence. Using a quartz waveguide and visible light in a waterenvironment, the evanescent field decays by approximately 90% within adistance of 100 nm from the waveguide/solution interface.

The same principles which create the evanescent field for lightpropagating in the waveguide or optical fiber allow the light which isgenerated when fluorophores luminesce to be captured back into theoptical element efficiently. Additionally, any light produced outsidethe evanescent zone is efficiently rejected from entering the opticalelement. The combination of these effects allows optical fibers orwaveguides to be used as efficient optical elements for measuring thepresence of and concentration of fluorophore labels on or near theirsurfaces in an aqueous environment. U.S. Pat. No. 4,447,546 describesone suitable method and apparatus for conducting fluorescenceimmunoassays employing an optical fiber to excite and measure evanescentzone fluorescence from labelled immunoreactant.

The invention can be applied to improve the sensitivity of fluorescentbinding assays using optical fibers or waveguides. The assay isperformed using reagents labelled with fluorescent moieties. Afterincubation of the particles, sample and reagents, the particles areconcentrated upon the surface of the waveguide or optical fiber. Becausethe surface area of the particles is greater than the geometric area ofthe waveguide or optical fiber, more fluorophores can be collected inthe evanescent zone surrounding the optical element. Hence, theluminescent signal from the particles will be larger and quantitation ofanalyte will be more sensitive, resulting in improved detection limits.

What is claimed is:
 1. An apparatus for performing a binding assay foran analyte of interest present in a sample based upon measurement ofelectrochemiluminescence at an electrode surface comprising:(a) a celldefining a sample containing volume intersecting with inlet and outletmeans (b) an electrode having a substantially horizontally positionedsurface exposed to and positioned below a portion of the samplecontaining volume; (c) means for impressing electrochemical energy uponsaid electrode sufficient to generate luminescence; (d) means formagnetically collecting particles along said surface; and (e) means formeasuring the luminescence generated at said electrode.
 2. An apparatusas defined in claim 1, wherein:said magnet comprises at least one magnetin north-south orientation positioned vertically below said electrode.3. An apparatus as defined in claim 2, wherein:said magnet comprises atleast one pair of magnets consisting of a first magnet and a secondmagnet that are separated by non-magnetic material.
 4. An apparatus asdefined in claim 3, wherein:said magnets are arranged in an antiparallelfashion whereby, for each pair of magnets, the north pole of the firstmagnet is proximate the south pole of the second magnet and the southpole of the first magnet is proximate the north pole of the secondmagnet.
 5. An apparatus as defined in claim 1 wherein:said magneticallycollecting means comprises a magnet that exerts a magnetic fieldcovering the region of space proximate said surface.
 6. An apparatus asdefined in claim 5, wherein:said magnet is located below said electrode.7. An apparatus as defined in claim 6 wherein:said magnet is capable ofbeing removed from its location beneath said electrode during theoperation of said measuring means.
 8. An apparatus as defined in claim5, wherein:said magnet comprises either a permanent magnet or anelectromagnet.
 9. An apparatus as defined in claim 8, wherein:saidmagnet is a permanent magnet.
 10. An apparatus as defined in claim 9,wherein:said magnet is an electromagnet.